Pyridazinone-substituted benzenesulfonamides display potent inhibition of membrane-bound human carbonic anhydrase IX and promising antiproliferative activity against cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2019.02.044

An expanded set of pyridazine-containing benzene sulfonamides was investigated for inhibition of four human carbonic anhydrase isoforms, which revealed a pronounced inhibition trend toward hCA IX, a cancer-related, membrane-bound isoform of the enzyme. Comparison of antiproliferative effects of these compounds against cancer (PANC-1) and normal (ARPE-19) cells at 50 μM concentration narrowed the selection of compounds to the eight which displayed selective growth inhibition toward the cancer cells. More detailed investigation in concentration-dependent mode against normal (ARPE-19) and two cancer cell lines (PANC-1 and SK-MEL-2) identified two lead compounds one of which displayed a notable cytotoxicity toward pancreatic cancer cells while the other targeted the melanoma cells. These findings significantly expand the knowledge base concerning the hCA IX inhibitors whose inhibitory potency against a recombinant enzyme translates into selective anticancer activity under hypoxic conditions which are aimed to model the environment of a growing tumor.

Full text of DOI:10.1016/j.ejmech.2019.02.044

Accepted Manuscript
Pyridazinone-substituted benzenesulfonamides display potent inhibition of
membrane-bound human carbonic anhydrase IX and promising antiproliferative
activity against cancer cell lines
Mikhail Krasavin, Anton Shetnev, Sergey Baykov, Stanislav Kalinin, Alessio
Nocentini, Vladimir Sharoyko, Giulio Poli, Tiziano Tuccinardi, Mikhail Korsakov,
Tatiana B. Tennikova, Claudiu T. Supuran
PII:
S0223-5234(19)30158-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.044
EJMECH 11136
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 January 2019
Revised Date: 10 February 2019
Accepted Date: 12 February 2019
Please cite this article as: M. Krasavin, A. Shetnev, S. Baykov, S. Kalinin, A. Nocentini, V. Sharoyko,
G. Poli, T. Tuccinardi, M. Korsakov, T.B. Tennikova, C.T. Supuran, Pyridazinone-substituted
benzenesulfonamides display potent inhibition of membrane-bound human carbonic anhydrase IX and
promising antiproliferative activity against cancer cell lines, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.044.
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ACCEPTED MANUSCRIPT
Pyridazinone-substituted benzenesulfonamides display potent inhibition of
membrane-bound human carbonic anhydrase IX and promising
antiproliferative activity against cancer cell lines
Mikhail Krasavin*, Anton Shetnev, Sergey Baykov, Stanislav Kalinin, Alessio Nocentini,
Vladimir Sharoyko, Giulio Poli, Tiziano Tuccinardi, Mikhail Korsakov, Tatiana B. Tennikova,
Claudiu T. Supuran*

ACCEPTED MANUSCRIPT
Pyridazinone-substituted benzenesulfonamides display potent inhibition of
membrane-bound human carbonic anhydrase IX and promising
antiproliferative activity against cancer cell lines
Mikhail Krasavina,*, Anton Shetnevb, Sergey Baykova, Stanislav Kalinina, Alessio Nocentinic,
Vladimir Sharoykoa, Giulio Polid, Tiziano Tuccinardid, Mikhail Korsakovb, Tatiana B.
Tennikovaa, Claudiu T. Supuranc,*
a Saint Petersburg State University, Saint Petersburg, 199034 Russian Federation
b The Ushinsky Yaroslavl State Pedagogical University, Yaroslavl 150000, Russian Federation
c Neurofarba Department, Universita degli Studi di Firenze, Florence, Italy
d Department of Pharmacy, University of Pisa, 56126 Pisa, Italy
*
Corresponding authors. E-mail addresses: m.krasavin@spbu.ru (M. Krasavin),
claudiu.supuran@unifi.it (C. T. Supuran)
ABSTRACT
An expanded set of pyridazine-containing benzene sulfonamides was investigated for inhibition
of four human carbonic anhydrase isoforms, which revealed a pronounced inhibition trend
toward hCA IX, a cancer-related, membrane-bound isoform of the enzyme. Comparison of
antiproliferative effects of these compounds against cancer (PANC-1) and normal (ARPE-19)
cells at 50 µM concentration narrowed the selection of compounds to the eight which displayed
selective growth inhibition toward the cancer cells. More detailed investigation in concentration-
dependent mode against normal (ARPE-19) and two cancer cell lines (PANC-1 and SK-MEL-2)
identified two lead compounds one of which displayed a notable cytotoxicity toward pancreatic
cancer cells while the other targeted the melanoma cells. These findings significantly expand the
knowledge base concerning the hCA IX inhibitors whose inhibitory potency against a
recombinant enzyme translates into selective anticancer activity under hypoxic conditions which
are aimed to model the environment of a growing tumor.
Keywords: carbonic anhydrase; isoform-selective inhibitors; periphery groups; primary
sulfonamides; subnanomolar inhibition; pyridazinone; phthalazinone, cancer cells; hypoxic
environment; growth inhibition assay.

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1. Introduction
The fundamental biochemical reaction of reversible hydration of carbon dioxide catalyzed by the
human carbonic anhydrases (hCAs) [1] produces bicarbonate anion and a proton and is,
therefore, an important regulatory mechanism for controlling the intra- and extracellular pH in
various part of the human body. Among the 15 hCA isoforms (each of them having different
cellular localization, catalytic activity and expression levels in normal vs. aberrantly functioning
cells [2]), many are already validated as targets for drug intervention while novel links to various
disease continue to be discovered [3]. Likewise, selective inhibition of pathogen CAs, not
interfering with the host hCA machinery, presents an attractive new option for antibiotic
development and is receiving increase attention. [4]. These new approaches to disease treatment
require that small molecules intended to produce a certain therapeutic effect are isoform-
selective and do not to affect the normal functioning of other isoforms, not involved in the
disease. Today, however, all clinically used carbonic anhydrase inhibitors (such as diuretics [5]
or drugs used to treat glaucoma-related intraocular hypertension [6]) are predominantly non-
selective as hCA inhibitors (Fig. 1).
Ki (nM)
hCA I
hCA II hCA IX hCA XII
acetazolamide
methazolamide
250
12
14
25
27
5.7
3.4
50
dorzolamide
brinzolamide
50,000
9
3
52
37
3.5
3.0
45,000
Fig. 1. Examples of clinically used, non-selective hCA inhibitors and their inhibition profile [7]
against the panel of human carbonic anhydrases (hCAs) interrogated in this work.
That being said, the landscape of investigational isoform-selective carbonic anhydrase inhibitors
(CAIs) has been widening. Noteworthy examples are isoxazole bis-sulfonamide inhibitor of

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membrane-bound hCA IV (1) [8], dual inhibitor of cytosolic hCA II and VII based on 2-
imidazoline scaffold (2) [9], selective biphenyl sulfonamide inhibitor of hCA XIV (3) [10], and
ring-opened N-alkyl saccharin derivative (4) selective for tumor-associated, membrane-bound
isoforms hCA IX and XII [11]. In the clinical phases of development, hCA IX-selective drug
SLC-0111 has now successfully completed phase I clinical trials for tumors overexpressing CA
IX [12] and is scheduled to enter phase II clinical trials in the near future. E7070 (indisulam) was
developed as novel antineoplastic therapy by Eisai Co., Ltd. It entered clinical development in
2005 and is currently in phase II clinical trials in the US and Europe [13]. Notably, from the
inhibition profile of SLC-0111 and, particularly, of E7070 it is evident that selectivity over the
cytosolic off-target hCA II isoform is not a pre-requisite to achieving clinical efficacy in cancer
treatment (Fig. 2).
Fig. 2. Examples of recently reported isoform-selective hCA inhibitors (1-4) and structures of
the most advanced hCA IX/XII-selective CAIs (SLC-0111 and E7070) currently in clinical trials
for cancer.
A metastatic tumor can survive and grow in the stressful hypoxic and acidic extracellular
environment surrounding it by overexpressing the membrane-bound hCA IX and hCA XII
isoforms [14] while inhibition of the latter can potentially retard the tumor growth and reduce its
size [15]. Today, mounting evidence has been gathered for tumors able to develop resistance to
various targeted small-molecule therapies [16]. In contrast, inhibition of hCA IX and hCA XII
overexpressed on the surface of tumor cells targets a fundamental mechanism of their survival, at

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the level of evolutionary primordial animatic machinery. Hence, the likelihood of resistance
emerging to drugs acting via this mechanism is considered low, which makes the latter an
attractive approach for small molecule intervention [17].
The majority of known CAIs are primary aromatic and heteroaromatic sulfonamides in which the
sulfonamide group acts as a metal binding motif and ensures affinity to the enzyme by
coordinating to its prosthetic Zn2+ ion [18], a binding mode universal for all hCA isoforms. It is
the molecular periphery of the inhibitor that can provide an additional binding to a particular
isoform and thus be the principal source of isoform-selective inhibition [19]. Thus exploration of
various periphery motifs for the pharmacophoric sulfonamide part of the prospective inhibitor
(via random screening or based on in silico modeling [20]) is especially significant for the
ongoing quest for selectively targeted CAIs.
In the course of exploring various substituted heterocyclic appendages for the CAI
pharmacophoric arene sulfonamide moiety (such as 1,3-oxazole [21], isoxazole [8], imidazoline
[9, 20], pyrazole [22] and 1,2,4-oxadiazole [23]), it came to our attention that pyridazinone-
substituted benzenesulfonamides 5 had been discovered and studied as potent inhibitors of
cytosolic hCA I and hCA II isoforms (as exemplified by compound 5a) [24]. Such a potent
inhibitory profile is perhaps unsurprising considering the almost ideal positioning of the polar
(pyridazinone carbonyl) and lipophilic (aromatic group) portions of the compound’s periphery
for cooperative contacts with the respective, distinctly delineated in hydrophobic character,
portions of the enzyme’s active site [19]. Considering this clear predisposition of representatives
of this chemotype to act as CAIs in general, we thought it important to prepare an expanded set
of analogs of pyridazinone-containing compounds and evaluate its inhibitory properties toward
the cancer-related, membrane-bound isoforms hCA IX and hCA XII. In addition to substituted
pyridazinone analogs 6 of the previously reported series [24], we aimed to explore their non-
aromatic analogs 7 as well as their differently substituted analogs 8 and benzene-fused series 9.
In all cases we expected the carbonyl group of the pyridazinone nucleus to effectively address
the hydrophilic side of the enzyme’s active site while the peripheral substituents would be
increasing the binding efficiency by interacting with the hydrophobic portion of the same (Fig.
3). Our ultimate goal was to identify compounds that display the clear propensity to inhibit the
hCA IX and/or hCA XII cell surface-bound isoforms (with or without inhibition of the cytosolic
off-targets hCA I and hCA II which are less important in the context of this study) and further
determine their potential to target cancer cells under hypoxic conditions (i. e. in the environment
where catalytic activity of these two isoforms becomes crucial for the cells’ survival and growth,
vide supra). Herein, we present the results of these studies.

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Fig. 3. Earlier reported pyridazinone-substituted benzenesulfonamides 5 (their design rationale
and CAI profile for a representative compound (5a)) and for series of analogs explored in this
work with respect to their hCA IX/hCA XII inhibition potential.
2. Results and discussion
2.1. Chemistry
A series of aromatic methyl ketones 10 were condensed with glyoxalic or pyruvic acid in
aqueous basic solution as described previously [25]. The intermediate compounds 11, without
isolation, were treated with an ethanolic solution of 4-hydrazinobenzenesulfonamide
hydrochloride salt (12) at reflux. This led to the conversion of the hydrazine component to the
free base form and subsequent condensation to form the pyridazinone ring and gave the target
compounds 6a-d in moderate to good yields (Scheme 1) [25].
Scheme 1. Synthesis of compounds 6a-d investigated in this work.
Compounds 7a-d belonging to the 5,6-dihydropyridazinone series were generated by similar
cyclocondensation between 12 and a series of commercially available γ-ketocarboxylic acids 13,
which provided the target compounds in good to excellent yields (Scheme 2) [26].
Scheme 2. Synthesis of compounds 7a-d investigated in this work.

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Similar condensation between 12 and mucochloric acid (14) yielded compound 8a in excellent
yield. Both chlorine atoms in 8a were removed by catalytic hydrogenation over 10% Pd on
carbon to give compound 8b in 79% yield [27]. At the same time, according to the literature data
[28], chlorine atom in position 5 of pyridazinone ring is more labile toward aromatic
nucleophilic substitution and thus it was replaced with a range of aliphatic amines in refluxing
ethanolic solution in the presence of triethylamine [29] to give compounds 8c-i in good to
excellent yields. Catalytic hydrogenation of a selected set of the latter provided compounds 8j-l
in high yield (Scheme 3).
Scheme 3. Synthesis of compounds 8a-l investigated in this work.
Finally, condensation of 12 with a small set of o-acylbenzoic acids 15 gave phthalazinone
compounds 9a-c in excellent yield (Scheme 4).
Scheme 4. Synthesis of compounds 9a-c investigated in this work.
2.2. Biochemical testing for CA inhibition
The twenty-three primary sulfonamide compounds 6a-d, 7a-d, 8a-l and 9a-c synthesized as
detailed above, were tested in CO2 hydration stopped-flow biochemical assay (see Experimental

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section) against two cytosolic (hCA I and II) and two membrane-bound, cancer-related hCA IX
and XII isoforms to produce the inhibition data (Ki) summarized in Table 1.
Table 1. Inhibitory profile of compounds 6a-d, 7a-d, 8a-k and 9a-c against hCA I, II, IX and
XII.a
Ki (nM)
Compound
R
Rʹ
hCA
II
hCA
IX
hCA
XII
hCA I
H
H
--
--
99.0
1.40
0.62
28.2
0.46
63.70
9.30
6a
6b
84.1
Me
H
--
--
100.5
227.4
2.90
4.8
2.30
50.8
8.80
8.70
6c
6d
Me
--
--
--
--
--
--
--
--
88.2
186.6
92.7
0.68
0.26
0.08
74.1
2.30
8.20
32.8
4.80
2.40
6.00
8.70
9.00
31.0
22.5
9.90
67.1
9.90
81.2
7a
7b
7c
7d
8a
8b
Ph
4-MeOC6H3
3,4-diClC6H3
Cl
--
--
5962
482.1
320.4
Cl
H
H
Cl
Cl
Cl
--
--
--
485.3
97.4
89.0
0.29
0.06
0.07
0.70
0.49
0.58
7.20
8.40
36.0
8c
8d
8e
Cl
Cl
--
--
738.4
95.1
60.5
0.09
89.6
0.67
85.5
9.40
8f
BnNH
8g

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Cl
--
481.5
0.67
2.80
10.1
8h
Cl
H
--
--
676.1
311.3
46.6
4.30
89.5
41.3
105.1
25.3
8i
8j
H
H
--
--
--
88.2
292.3
72.3
0.08
0.55
0.23
0.56
2.10
0.32
6.30
9.80
50.6
8k
8l
--
--
9a
--
--
90.7
89.7
3.50
0.03
8.90
0.24
66.4
9.00
9b
9c
Me
a Mean Ki values from 3 different stopped-flow assays (errors were in the range of ±5-10% of the
reported values).
From a brief examination of the data presented in Table 1, it becomes evident that in general, the
compounds prepared and investigated in this work retained subnanomolar to low nanomolar
inhibitory potency against cytosolic hCA II isoform while the potency toward hCA I (also a
cytosolic form) decrease dramatically in comparison to the earlier reported data (cf. compound
5a). At the same time, the inhibitory potency toward cancer-related hCA IX was established to
reach into the subnanomolar range, thereby making this diverse series of compounds worthy
further investigation as antiproliferative agents for cancel cells under hypoxic conditions.
Particularly encouraging is a concomitant inhibition of hCA XII isoform, albeit with surprisingly
flat SAR (i.e., the single- to double-digit nanomolar inhibition of this isoform appears insensitive
to variations in the inhibitor structure). It should be noted that inhibition of hCA II, in general,
does not present a significant obstacle to antitumor efficacy (as evidenced by the advanced status
of compound E7070 (indisulam), also a dual hCA II/IX inhibitor, currently in clinical trial for
cancer [13]). Moreover, considering the vastly different cellular localization of these two
isoforms, the de facto selectivity will be governed in the end by the potentially higher
extracellular vs. intracellular concentration of the inhibitor [30].

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Notable SAR facts that surface from the data presented in Table 1, include the relative
insensitivity of the inhibitory profile to the substitutions around the flat pyridazinone core as far
as large substituents in position 4 are concerned. Indeed, the profile of compounds 6a-d is nearly
identical to that of bis-chloro compound 8a and unsubstituted compound 8b. At the same time, in
the non-flat, 5,6-dihydropyridone series, hCA II potency appears quite sensitive to the nature of
the aromatic group (cf. compounds 7c and 7d displaying a ~1000-fold drop in inhibitory
activity). Similarly sensitive to the substitution pattern appears the phthalazinone series where
introduction of a methyl group in compound 9c improved the hCA II potency 10-fold compared
to compounds 9a. However, the dimethoxy substitution in compound 9b appears to be
detrimental to both hCA II and hCA IX inhibition. In the amino-substituted pyridazinone series,
only tetrahydroisoquinoline substitution in 8f appears to lead to poorer inhibition (compared, for
instance, to its ‘disconnected’ analog 8g) of both hCA II and hCA IX. The inhibitory profile of
the chlorinated amino-substituted compounds looks similar to that of their des-chloro
counterparts, except for the pair 8c/8j where removal of the chlorine atom lead to a 10-fold drop
in hCA II and hCA IX potency.
2.3. Docking studies
In order to provide rational interpretations of the SAR data derived from the experimental
results, representative compounds belonging from the different series of synthesized derivatives
were studies through a molecular modeling analysis comprising a robust docking procedure
followed by energy minimization in explicit water environment (see Experimental section for
details). The phthalazinone derivatives 9a and 9c, which showed subnanomolar potency against
hCA II and hCA IX, were docked into the catalytic site of the two enzymes with the aim of
evaluating their potential binding mode. Compound 9a was predicted to chelate the prosthetic
zinc ion of hCA II through the sulfonamide group, which also interacts with T198 forming H-
bonds with both the backbone nitrogen and the hydroxyl group of the residue (Fig. 4A). The
phenylene linker of the ligand shows van der Waals interactions with H94 and Q92 from one
side and takes hydrophobic contacts with V121, L140 and especially L197 from the other side.
Finally, the phthalazinone moiety is placed into the narrow space delimited by I91, Q92, V121
and F130, forming a strong π-π stacking with F130 and an H-bond with the side chain of Q92
established through its carbonyl oxygen. The binding mode generated for 9a into hCA IX is very
similar to the disposition predicted into hCA II, in agreement with the comparable activity of the
ligand toward the two enzyme isoforms (Fig. 4B). In fact, compound 9a forms the same pattern
of H-bonds and lipophilic interactions with the homolog residues of hCA IX, with the only
exception of the π-π stacking interaction that is absent due to the presence of V262 in place of

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the non-conserved residue F130 (hCA II). However, the ligand is still able to form strong van der
Waals interactions with the flat hydrophobic wall of hCA IX constituted by L223, V262 and
L266.
The increase in hCA II inhibitory activity obtained by the introduction of a methyl group in
compound 9c seems to be due to improved hydrophobic interactions and a stabilization of the
binding pose, compared to 9a. In fact, the methyl group of 9c, well fits a small hydrophobic
pocket delimited by F130, V134, I197 and P201, formed by the protrusion of F130 side chain
towards the entrance of the binding site. The interaction into this pocket contributes to lock the
ligand into its binding disposition, besides allowing additional lipophilic contacts with the pocket
residues (Fig. 4C). Interestingly, compound 9c maintains the H-bond with Q92 and a T-shaped
stacking interaction with F130. The observation that no potency improvement against hCA IX is
obtained for 9c with respect to 9a is in agreement with the absence of the small hydrophobic
pocket within hCA IX binding site. On the contrary, the hCA II potency drop obtained by the
introduction of the two methoxy groups in compound 9b can be rationalized by considering the
steric bulk of the two substituents, which would prevent the ligand to be placed into the narrow
space between Q92 and F130. In fact, compound 9b is predicted to dispose the phthalazinone
core perpendicularly with respect to the binding mode of 9a, in order to avoid steric hindrance
with the surrounding residues (Fig. 4D). However, this is paid with the loss of the H-bond with
Q92, which is consistent with the reduced activity of the ligand. Similar considerations can be
also valid for justifying the activity of the ligand against hCA IX, which is comparable to that
shown against hCA II.
Among the amino-substituted pyridazinone derivatives, compound 8c showed subnanomolar
potency against both hCA II and hCA IX, while its des-chloro analogue 8j was found to have
reduced activity. The predicted binding mode of the two compounds into hCA II is similar and
consistent with the binding disposition hypothesized for compound 9a (Fig. 4A). In fact, besides
the sulfonamide groups, also the phenylene linker and the phthalazinone ring of 8c and 8j
present an orientation comparable with that observed for 9a, forming a similar pattern of
interactions with the surrounding protein residues, included the H-bond with the side chain of
Q92 (Fig. 5). However, the π-π stacking with F130 is not shown due to the absence of the fused
phenyl ring in 8c and 8j, but the positively charged piperazine groups of the two ligands are able
to form a water-bridged interaction with E69. The positive effect on ligand potency determined
by the chlorine atom could be due to the additional hydrophobic contacts and a better shape
complementarity with the protein observed in compound 8c with respect to 8j. In fact, the
chlorine atom can form extensive lipophilic interactions with I91, Q92, V121 and F130 that can

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also compensate for the lack of π-π stacking with F130 and stabilize the ligand disposition into
the protein catalytic site, thus justifying the higher potency of 8c compared to 8j. The reduced
activity of both ligands against hCA IX can be ascribed to the fact that E69 of hCA II is a non-
conserved residue replaced by a threonine in hCA IX; therefore, the two ligands cannot form the
water-bridged interaction with E69. However, while 8c shows only a slight (2.5-fold) decrease in
potency, compound 8j presents a 10-fold lower activity on hCA IX compared to hCA II: this
data is consistent with the binding mode proposed for the two derivatives, considering the less
direct interaction with E69 predicted for 8c (mediated by two water molecules instead of one as
in 8j) and the presence of the chlorine atom, which can reduce the impact of the missing
interaction with E69, compared to 8j.
Fig. 4. Predicted binding mode of compound 9a into hCA II (A) and hCA IX (B); predicted
binding mode of compound 9c (C) and 9b (D) into hCA II. Ligand-protein H-bonds are shown as
black dashed lines, the protein surface in the proximity of the ligands is shown in grey.
Finally, a different water-bridged interaction could contribute to the different hCA II inhibitory
potency observed for the non-flat 5,6-dihydropyridone derivatives 7c and 7d. Compound 7c,
which showed picomolar potency against hCA II, was predicted to assume a binding mode in

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line with those calculated for the other hCAIs herein analyzed. Interestingly, despite the
distortion introduced in the core ring due to the lack of aromaticity, the ligand is still able to form
an H-bond with Q92 and extensive hydrophobic interactions with F130. Moreover, although the
methoxyphenyl group of the ligand is highly solvent exposed, the water-mediated H-bond with
W5 can counterbalance the unfavorable desolvation effect and positively contribute to the ligand
potency. On the contrary, the double chlorinated analogue 7d is not able to form a similar
interaction and it was predicted to orient its dichlorophenyl ring differently, in order to prevent
any steric hindrance with W5 and to maximize the lipophilic interactions with both this and other
residues such as H64 and N67. However, this different disposition also prevents the formation of
the H-bond with Q92, thus contributing to explain the considerable reduction in hCA II potency
of 7d with respect to 7c (Fig. 6).
Fig. 5. Predicted binding mode of compound 8c (A) and 8j (B) into hCA II (A). Ligand-protein
H-bonds are shown as black dashed lines, the protein surface in the proximity of the ligands is
shown in grey.
Fig. 6. Predicted binding mode of compound 7c (A) and 7d (B) into hCA II. Ligand-protein H-
bonds are shown as black dashed lines, the protein surface in the proximity of the ligands is
shown in grey.

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2.4. Antiproliferative activity against normal and cancer cell lines
The potent inhibition of membrane-bound, cancer-related enzyme hCA IX by virtually all
compounds 6a-d, 7a-d, 8a-l and 9a-c (some of them reaching into the subnanomolar potency
range), on top of fairly promising inhibition profile with respect to hCA XII, clearly warranted
their further investigation for antiproliferative effects in cancer cells. As we already mentioned
the absence of selectivity between these isoforms and also against hCA II should not be
perceived as a substantial disadvantage, considering the fact the vastly different cellular
localization of these targets and, as a result, the potential for gaining selectivity by suppressing
cell membrane permeability in the course of subsequent lead optimization [30]. While evaluation
of the antiproliferative profile for cancer cells in comparison to normal cells should be performed
under normoxic as well as hypoxic conditions, sulfonamide CAs are known to be more likely to
exert their cytotoxicity profile under hypoxic conditions, i. e. those that closely reproduce the
environment of a growing solid tumor, particularly with respect to pH disbalance and
overexpression of hCA IX and hCA XII isoforms [31].
Fig. 7. Cell viability MTT assay results for compounds 6a-d, 7a-d, 8a-l and 9a-c (50 µM)
against APRE-19 and PANC-1 cell lines (values are expressed as the mean ± SEM of three
experiments: (∗) P < 0.05 and (∗∗) P < 0.01 in comparison to control (0 µM).
Compounds 6a-d, 7a-d, 8a-l and 9a-c were screened at 50 µM concentration for their ability to
affect the cell culture viability of non-cancerous human retinal pigment epithelial cell line
ARPE-19 [32] as well as pancreas ductal adenocarcinoma cell line PANC-1 [33]. The initial
testing was performed relative to control (0 µM of the test compounds) under chemically
induced (CoCl2) hypoxia [34]. As it is evident from the screening results presented in Fig. 7,
cobalt(II) chloride itself did not affect the number of cells in the culture. Compound 6d, for some

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reason, displayed a pronounced cytotoxicity toward the normal as well as cancer cells and thus
was excluded from further consideration. However, a number of compounds (particularly, eight
compounds 7a, 7c, 8a-e and 8j) proved to be >30% more cytotoxic towards PANC-1 vs. ARPE-
19 cell line under the chemically induced hypoxia conditions and were thus perceived as leads
worthy further characterization.
7a
7c
8a
8b
8c
8d
8e
8j
Fig. 8. Cell viability MTT assay results for compounds 7a, 7c, 8a-e and 8j (30 µM, 100 µM and
300 µM) against APRE-19, PANC-1 and MEL cell lines (values are expressed as the mean ±
SEM of three experiments: (∗) P < 0.05 and (∗∗) P < 0.01 in comparison to control (0 µM, not
shown).

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These nine frontrunner compounds were tested in the MTT cell viability assay at three different
concentrations (30 µM, 100 µM and 300 µM) against normal (ARPE-19) as well as two cancer
cell lines (PANC-1 and also melanoma SK-MEL-2 [35] cell line), this time grown under
normoxic as well as chemically induced hypoxic conditions. The antiproliferative profiles
displayed by compounds 7a, 7c, 8a-e and 8j under this testing scheme are shown in Fig. 8.
Compound 8a displayed a pronounced, non-selective cytotoxicity toward normal as well as both
cancer cell lines. This behavior is likely a consequence of the compound’s reactivity toward
nucleophilic moieties which results in non-specific covalent modification of biomolecules.
Similarly, compounds 7c and 8d displayed a dose-dependent cytotoxicity against ARPE-19 cells
and thus were not considered further. Compounds 8b and 8c displayed no particular cytotoxicity
neither to normal nor to cancerous cells, even at high concentration. Only at 300 µM
concentration, compound 8j appears selectively cytotoxic to melanoma cells. However, the main
result of these comprehensive experiments, is that compound 8e can be viewed as a promising
cytotoxic agent for melanoma cells (under either hypoxic or normoxic conditions where it dose-
dependently inhibited the growth of cancer cells but not normal cells) while compound 7a was
selectively cytotoxic to pancreatic cancer cell under hypoxic conditions. It is noteworthy that
these agents belong to different structural classes and could be identified only via the ‘screening
funnel’ scheme implemented in this work. It should be duly noted that in general, modeling of
antitumor effects of hCA IX inhibitors by antiproliferative effects against a disperse cell culture,
even grown under hypoxic conditions, is exceedingly difficult and requires cell viability
screening at relatively high compound concentrations [31]. As the result, potent inhibition of
hCA IX determined in a biochemical assay may not translate directly into similarly promising
antiproliferative activity in vitro. This mandates that advanced candidates possessing promising
antiproliferative profiles are selected from hCA IX based on preliminary cell viability testing and
not only based on best Ki values.
3. Conclusion
By preparing and investigating an extended set of pyridazine-containing benzene sulfonamides,
we established a pronounced propensity of this class of compounds to inhibit cancer-related,
membrane bound isoform IX of human carbonic anhydrase (hCA IX), an observation that had
not been made in the previous studies due to a limited number of isoforms included in the testing
panel. This discovery prompted us to investigate the effects of these compounds on cancerous vs.
normal cells under chemically induced hypoxia (i. e. the conditions rendering the catalytic

ACCEPTED MANUSCRIPT
activity of hCA IX crucial survival mechanism of cancer cells. To our delight, eight compounds
displayed a noticeable window between cytotoxicity against the normal (ARPE-19) and cancer
(PANC-1) cells which warranted their further investigation. Testing these compounds at three
different concentrations (30 µM, 100 µM and 300 µM) against ARPE-19 cells in comparison
with PANC-1 and SK-MEL-2 cells revealed that one of the compounds (7a) displayed
pronounced cytotoxicity toward pancreatic cancer cells while the other (8e) clearly targeted
melanoma cells. The observed structure-activity relationships were analyzed by docking the key
cornerstone compounds into the crystal structure of different isoforms, which allowed
rationalizing the observed SAR trends. Altogether, these findings significantly expand the range
of known hCA IX inhibitors whose biochemical activity translates (albeit indirectly) into the
selective antiproliferative activity against cancer cells under hypoxic conditions that model the
environment of a growing tumor.
4. Experimental section
4.1. General experimental
All and reagents and solvents were obtained from commercial sources and used without
purification. All reactions implemented in an open flask without any protection from CO2 and
H2O. Reactions were monitored by analytical thin layer chromatography (TLC) Macherey-
Nagel, TLC plates Polygram® Sil G/UV254. Visualization of the developed chromatograms was
1
13
performed by fluorescence quenching at 254 nm. H and C NMR spectra were measured on
1
13
Bruker AVANCE DPX 400 (400 MHz for H and 100 MHz for C respectively). All chemical
shifts (δ ppm ) are given in parts per million (ppm) with reference to solvent residues in DMSO-
d6-d6 (2.50 for proton and 39.52 for carbon) and coupling constant (J) are reported in hertz (Hz).
Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, br = broad. Melting points were determined on Electrothermal IA 9300 series Digital
Melting Point Apparatus. Mass spectra were recorded on microTOF spectrometers (ESI
ionization). Analytical HPLC was carried out on Shimadzu LC-20AP chromatograph equipped
with spectrophotometric detector and Supelco Discovery C18, 5µm, 15cm×3mm column. Mobile
phase: (A) 0.1% TFA in water – (B) 0.1% TFA in acetonitrile; gradient: 5−95% B (0 − 15min),
95% B (15 − 20min); flow rate 1 mL/min, temperature – 40 °C, detection UV at 214 and 254
nm. Injection volume 20µl. All compounds tested in biology assays were judged to be at least
95% pure by analytical HPLC.
4.2. Synthetic organic chemistry

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General Procedure (GP1) for the synthesis of compounds 6a-d, 7a-d, 8a, 9a-c
A mixture of appropriate 1,4 - dicarbonyl compound 11, 13, 14 or 15 (0.001 mol) and 4-
hydrazinobenzenesulfonamide hydrochloride 12 (0.001 mol) in ethanol (10–40 mL) was refluxed
for 8-24 h. The reaction mixture was evaporated to give a crude solid. Crude product was stirred
with 5-10 % sodium bicarbonate solution (25 mL) for 1h. It was filtered, washed with 2% acetic
acid and then with water. It was dried and crystallized from ethanol to give (6a-d, 7a-d, 8a, 9a-
c).
4-(3-(Benzo[d][1,3]dioxol-5-yl)-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (6a)
1
Yield 174 mg (47%). Beige solid, m.p. 281-281,5°С, H NMR (400 MHz, DMSO-d6) δ ppm
8.12 (d, J= 9.8 Hz, 1H), 7.95 (q, J= 8.7 Hz, 4H), 7.54 – 7.43 (m, 4H), 7.18 (d, J= 9.8 Hz, 1H),
13
7.04 (d, J= 8.1 Hz, 1H), 6.11 (s, 2H). C NMR (101 MHz, DMSO-d6) δ ppm 158.95, 149.20,
148.59, 144.76, 144.46, 143.60, 131.77, 131.58, 128.65, 126.64, 126.41, 121.23, 109.01, 106.61,
102.09. HRMS (ESI, m/z): calculated for C17H13N3O5S [M+H]+ 372.0649; found 372.0643.
4-(3-(2-Hydroxyphenyl)-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (6b)
1
Yield 165 mg (48 %). White solid, m.p. 269-270 °С. H NMR (400 MHz, DMSO-d6) δ ppm
10.38 (s, 1H), 8.01 (d, J= 9.8 Hz, 1H), 7.97 (d, J= 8.6 Hz, 2H), 7.91 (d, J= 8.6 Hz, 2H), 7.58 (d,
J= 7.6 Hz, 1H), 7.56 - 7.33 (br. s, 2H), 7.31 (t, J= 7.7 Hz, 1H), 7.14 (d, J= 9.8 Hz, 1H), 7.00 (d,
J= 8.1 Hz, 1H), 6.91 (t, J= 7.5 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) 159.00, 156.05, 145.90,
144.45, 143.66, 135.15, 131.54, 130.10, 130.05, 126.66, 126.50, 121.84, 119.89, 116.95, 130.05,
126.66, 126.50, 121.84, 119.89, 116.95. HRMS (ESI, m/z): calculated for C16H13N3O4S [M+H]+
344.0700; found 344.0696.
4-(5-Methyl-6-oxo-3-(pyridin-3-yl)pyridazin-1(6H)-yl)benzenesulfonamide (6c)
1
Yield 154 mg (45 %). Light brown solid, m.p. 128-129 °С. H NMR (400 MHz, DMSO-d6) δ
ppm 9.87 (s, 1H), 9.02 (s, 1H), 8.53 (s, 1H), 8.17 (s, 1H), 7.70 (s, 2H), 7.38 (s, 3H), 7.11 (s, 2H),
13
2.33 (s, 3H). C NMR (101 MHz, DMSO-d6) δ ppm 149.06, 148.75, 147.17, 141.29, 134.83,
134.49, 133.21, 127.74, 123.88, 112.67, 13.39. HRMS (ESI, m/z): calculated for C16H14N4O3S
[M+H]+ 342.0787; found 342.0789.
4-(6-Oxo-3-(pyridin-4-yl)pyridazin-1(6H)-yl)benzenesulfonamide (6d)
1
Yield 283 mg (86 %). Orange solid, m.p. 282-283°С. H NMR (400 MHz, DMSO-d6) δ ppm
8.72 (dd, J= 4.6, 1.5 Hz, 2H), 8.25 (d, J= 9.8 Hz, 1H), 8.02 – 7.96 (m, 2H), 7.96 – 7.89 (m, 4H),
7.51 (s, 2H), 7.30 (d, J= 9.8 Hz, 1H). 13C (101 MHz, DMSO-d6) δ ppm 159.18, 150.94, 144.24,

ACCEPTED MANUSCRIPT
144.00, 142.89, 141.62, 131.89, 131.41, 126.74, 126.61, 120.65. HRMS (ESI, m/z): calculated
for C15H12N4O3S [M+H]+ 329.0703; found 329.0696.
4-(3-Methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl)benzenesulfonamide (7a)
1
Yield 147 mg (55 %). white crystal, m.p. 145-147 °С. H NMR (400 MHz, DMSO-d6) δ ppm
ppm 8.28 (d, J= 8.4 Hz, 2H), 8.04 (d, J= 8.4 Hz, 2H), 7.60 (s, 2H), 3.17 (m, 1H), 1.33 (d, J= 6.9
Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ ppm 165.96, 156.97, 144.12, 141.26, 126.31,
124.55, 27.75, 26.14, 23.34. HRMS (ESI, m/z): calculated for С11H14N3O3S [M+H]+ 268.0751;
found 268.0754.
4-(6-Oxo-3-phenyl-5,6-dihydropyridazin-1(4H)-yl)benzenesulfonamide (7b)
Yield 221 mg (67 %). White solid, m.p. 254-255 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.93
– 7.84 (m, 4H), 7.81 (d, J= 8.5 Hz, 2H), 7.52 – 7.45 (m, 3H), 7.39 (s, 2H), 3.17 (t, J= 8.0 Hz,
13
2H), 2.78 (t, J= 8.0 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ ppm 166.17, 153.38, 144.29,
141.58, 135.75, 130.50, 129.10, 126.71, 126.44, 124.84, 27.93, 22.77. HRMS (ESI, m/z):
calculated for C16H15N3O3S [M+H]+ 330.0907; found 330.0903.
4-(3-(4-Methoxyphenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)benzenesulfonamide (7c)
Yield 238 mg (66 %). White solid, m.p. 198-199 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.84
(m, 6H), 7.37 (s, 2H), 7.03 (d, J= 8.8 Hz, 2H), 3.82 (s, 3H), 3.13 (t, J= 8.0 Hz, 2H), 2.75 (t, J=
13
8.0 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ ppm 166.19, 161.27, 153.22, 144.36, 141.42,
128.38, 128.09, 126.40, 124.71, 114.48, 55.80, 28.04, 22.63. HRMS (ESI, m/z): calculated for
C17H17N3O4S [M+H]+ 360.1013; found 360.1014.
4-(3-(3,4-Dichlorophenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)benzenesulfonamide (7d)
1
Yield 194 mg (94 %). White solid, m.p. 267-268 °С. H NMR (400 MHz, DMSO-d6) δ ppm
8.06 (d, J= 2.1 Hz, 1H), 7.91 – 7.87 (m, 2H), 7.85 (dd, J= 8.5, 2.1 Hz, 1H), 7.80 – 7.75 (m, 2H),
7.74 (d, J= 8.5 Hz, 1H), 7.38 (s, 2H), 3.18 (t, J= 8.1 Hz, 2H), 2.79 (t, J= 8.1 Hz, 2H). 13C NMR
(101 MHz, DMSO-d6) δ ppm 165.99, 151.14, 144.08, 141.82, 136.43, 132.98, 132.06, 131.28,
128.47, 126.87, 126.48, 125.08, 27.66, 22.66. HRMS (ESI, m/z): calculated for C16H13Cl2N3O3S
[M+H]+ 398.0128; found 398.0123.
4-(4,5-Dichloro-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (8a)
Yield 269 mg (96 %). White solid, m.p. 258-259 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.39
(s, 1H), 7.98 (d, J= 8.5 Hz, 2H), 7.79 (d, J= 8.5 Hz, 2H), 7.50 (s, 2H). 13C NMR (101 MHz,

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DMSO-d6) δ ppm 156.10, 144.43, 143.72, 137.38, 136.58, 134.58, 126.83, 126.77. HRMS (ESI,
m/z): calculated for C10H7Cl2N3O3S [M+H]+ 319.9658; found 319.9654.
4-(1-Oxophthalazin-2(1H)-yl)benzenesulfonamide (9a)
Yield 263 mg (87 %). White solid, m.p. 247-248 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.63
(s, 1H), 8.35 (d, J= 7.8 Hz, 1H), 8.02 (d, J= 6.2 Hz, 2H), 7.95 (m, 3H), 7.87 (d, J= 8.6 Hz, 2H),
7.48 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ ppm 158.85, 144.72, 143.25, 139.87, 134.67,
133.05, 129.67, 128.05, 127.64, 126.77, 126.68, 126.60. HRMS (ESI, m/z): calculated for
С13H11N4O3S [M+H]+ 303.0546; found 303.0531.
4-(7,8-Dimethoxy-1-oxophthalazin-2(1H)-yl)benzenesulfonamide (9b)
Yield 326 mg (90 %). White solid, m.p. 263-264 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.40
(s, 1H), 7.94 (d, J= 8.3 Hz, 2H), 7.81 (d, J= 8.3 Hz, 2H), 7.76 (s, 2H), 7.47 (s, 2H), 3.97 (s, 3H),
13
3.81 (s, 3H). C NMR (101 MHz, DMSO-d6) δ ppm 156.94, 156.14, 148.37, 145.14, 142.95,
139.31, 126.88, 126.47, 124.62, 124.46, 121.96, 119.90, 61.57, 56.92. HRMS (ESI, m/z):
calculated for C16H15N3O5S [M+H]+ 362.0805; found 362.0819.
4-(4-Methyl-1-oxophthalazin-2(1H)-yl)benzenesulfonamide (9c)
Yield 243 mg (77 %). White solid, m.p. 257-258 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.37
(d, J= 7.8 Hz, 1H), 8.05 – 8.00 (m, 2H), 7.98 – 7.91 (m, 3H), 7.90 – 7.85 (m, 2H), 7.46 (s, 2H),
13
2.62 (s, 3H). C NMR (101 MHz, DMSO-d6) δ ppm 158.74, 145.34, 144.74, 143.06, 134.52,
132.73, 129.64, 127.88, 127.16, 126.57, 126.43, 19.16. HRMS (ESI, m/z): calculated for
C15H14N3O3S [M+H]+ 316.0750; found 316.0762.
General Procedure (GP2) for the synthesis of compounds 8c-f, 8i-h
A
mixture of 4-(4,5-dichloro-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (1 mmol),
triethylamine (0.15 ml, 1,1 mmol) and 1,05 mmol the appropriated amine in ethanol (15-25 ml)
was heated under reflux for 24 hours. After cooling the precipitated solids were collected,
washed with water, ethanol and dried to afford the title compound without any additional
purification.
4-(5-Chloro-4-(4-ethylpiperazin-1-yl)-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (8с)
1
Yield 490 mg (79 %). White solid, m.p. 261-261,5°С. H NMR (400 MHz, DMSO-d6) δ ppm
8.15 (s, 1H), 7.93 (d, J= 8.7 Hz, 2H), 7.75 (d, J= 8.7 Hz, 2H), 7.46 (s, 2H), 3.56 – 3.47 (t, J= 4.9
13
Hz, 4H), 2.39 (q, J= 7.2 Hz, 2H), 1.04 (t, J= 7.2 Hz, 3H). C NMR (101 MHz, DMSO-d6) δ

ACCEPTED MANUSCRIPT
ppm 157.52, 147.63, 144.24, 143.56, 133.66, 126.63, 126.34, 113.62, 52.81, 51.97, 49.00, 12.29.
HRMS (ESI, m/z): calculated for C16H20ClN5O3S [M+H]+ 398.1048; found 398.1049.
4-(4-(Azepan-1-yl)-5-chloro-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (8d)
Yield 353 mg (92 %). White solid, m.p. 207-208 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.10
(s, 1H), 7.92 (d, J= 8.6 Hz, 2H), 7.76 (d, J= 8.6 Hz, 2H), 7.45 (s, 2H), 3.78 (t, J= 5.9 Hz, 4H),
1.79 (s, 4H), 1.57 (s, 4H). 13C NMR (101 MHz, DMSO-d6) δ ppm 157.61, 146.01, 144.33,
143.23, 132.11, 126.54, 126.08, 106.58, 52.10, 28.76, 26.50. HRMS (ESI, m/z): calculated for
C16H19ClN4O3S [M+H]+ 383.0939; found 383.0937.
4-(5-Сhloro-4-morpholino-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (8e)
Yield 315 mg (85 %). White solid, m.p. 255-256 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.16
(s, 1H), 7.93 (d, J= 8.7 Hz, 2H), 7.76 (d, J= 8.7 Hz, 2H), 7.46 (s, 2H), 3.79 – 3.69 (m, 4H), 3.59
– 3.47 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ ppm 157.50, 147.60, 144.21, 143.61, 133.59,
126.65, 126.35, 114.22, 66.62, 49.35. HRMS (ESI, m/z): calculated for C14H15ClN4O4S [M+H]+
371.0575; found 371.0562.
4-(5-Сhloro-4-(3,4-dihydroisoquinolin-2(1H)-yl)-6-oxopyridazin-1(6H)-yl)benzenesulfonamide
(8f)
Yield 190 mg (49 %). White solid, m.p. 283-284 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.23
(s, 1H), 7.94 (d, J= 8.6 Hz, 2H), 7.77 (d, J= 8.6 Hz, 2H), 7.47 (s, 2H), 7.22 (s, 4H), 4.76 (s, 2H),
3.81 (s, 2H), 3.01 (s, 2H). 13C NMR NMR (101 MHz, DMSO-d6) δ ppm 157.58, 147.64, 144.27,
143.53, 134.57, 133.72, 133.19, 129.20, 127.14, 126.74, 126.64, 126.61, 126.31, 112.51, 50.88,
47.34, 28.99. HRMS (ESI, m/z): calculated for C19H18ClN4O3S [M+H]+ 417.0783; found
417.0776.
4-(5-Сhloro-6-oxo-4-(4-(pyridin-2-yl)piperazin-1-yl)pyridazin-1(6H)-yl)benzenesulfonamide (8i)
Yield 340 mg (81 %). Beige solid, m.p. 302-304°С. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.18
(d, J= 15.0 Hz, 2H), 7.95 (d, J= 7.5 Hz, 2H), 7.78 (d, J= 7.4 Hz, 2H), 7.53 (d, J= 41.2 Hz, 3H),
6.89 (d, J= 7.9 Hz, 1H), 6.70 (s, 1H), 3.66 (s, 8H). 13C NMR (101 MHz, DMSO-d6) δ ppm
159.18, 157.53, 148.08, 147.69, 144.24, 143.58, 138.14, 133.66, 126.66, 126.33, 113.87, 107.74,
48.64, 45.19. HRMS (ESI, m/z): calculated for C19H20ClN6O3S [M+H]+ 447.1001; found
447.1015.
4-(4-(Benzylamino)-5-chloro-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (8g)

ACCEPTED MANUSCRIPT
Yield 207 mg (53 %). White solid, m.p. 225-226 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm
8.01 (s, 1H), 7.89 (d, J= 8.5 Hz, 2H), 7.72 (d, J= 8.5 Hz, 2H), 7.66 (t, J= 6.4 Hz, 1H), 7.44 (s,
2H), 7.38 (d, J= 4.3 Hz, 4H), 7.29 (m, 1H), 4.67 (d, J= 6.4 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) δ ppm 156.71, 144.89, 144.33, 143.30, 139.42, 129.12, 129.00, 127.69, 127.32,
126.49, 126.33, 105.34, 45.69. HRMS (ESI, m/z): calculated for C17H15ClN4O3S [M+H]+
391.0626; found 391.0619.
4-(5-Chloro-4-((3-morpholinopropyl)amino)-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (8h)
1
Yield 294 mg (69 %). White solid, m.p. 196 -197 °С. H NMR (400 MHz, DMSO-d6) δ ppm
8.18 (s, 1H), 7.91 (d, J= 8.0 Hz, 2H), 7.74 (d, J= 7.9 Hz, 2H), 7.45 (s, 2H), 7.14 (s, 1H), 3.69 –
3.42 (m, 6H), 2.37 (s, 6H), 1.75 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ ppm 156.69, 145.02,
144.44, 143.22, 129.21, 126.52, 126.27, 104.36, 66.55, 56.30, 53.77, 41.74, 26.20. HRMS (ESI,
m/z): calculated for C17H22ClN5O4S [M+H]+ 428.1154; found 428.1159.
General procedure 3 (GP3) of hydrogenation of 8a and 8c-i
The chloro compound (8a or 8c-i) (1 mmol) was suspended in 20 mL of methanol with the
addition of (2,1 or 1,1 mmol respectively) of triethylamine. After the mixture was purged with
inert atmosphere, 5 mg of 10 % palladium on carbon catalyst (2-5 % by weight of 8a or 8c-i)
was added, and the mixture was treated with hydrogen gas at 5 bar in a Parr hydrogenation
apparatus for 16 h at 50 °C. The hot mixture was filtered through a Celite pad, and the pad was
washed with 30 mL of boiling methanol. The filtrates were combined and evaporated in vacuo to
dryness at 50 °C. The residue was triturated with water and filtered to yield of crude product.
Recrystallization of a small sample from ethanol yielded an analytical sample.
4-(6-Oxopyridazin-1(6H)-yl)benzenesulfonamide (8a)
Yield 199 mg (79 %). white solid, m.p. 170-172 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.20
– 7.70 (m, 5H), 7.48 (s, 3H), 7.12 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ ppm 159.74,
144.28, 143.75, 138.51, 133.12, 131.35, 126.66, 126.46. HRMS (ESI, m/z): calculated for
C10H9N3O3S [M+H]+ 252.0437; found 252.0449.
4-(4-(4-Ethylpiperazin-1-yl)-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (8c)
1
Yield 233 mg (64 %). Beige solid, m.p. 221-222 °С. H NMR (400 MHz, DMSO-d6) δ ppm
8.24 (d, J= 2.7 Hz, 1H), 7.88 (d, J= 8.7 Hz, 2H), 7.75 (d, J= 8.7 Hz, 2H), 7.41 (s, 2H), 5.93 (d,
J= 2.6 Hz, 1H), 3.56 – 3.38 (m, 4H), 2.48 – 2.41 (m, 4H), 2.37 (q, J= 7.2 Hz, 2H), 1.03 (t, J=
12.9, 5.8 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ ppm 160.36, 149.95, 144.37, 142.78,

ACCEPTED MANUSCRIPT
131.76, 126.42, 125.82, 100.02, 52.13, 51.92, 46.14, 12.36. HRMS (ESI, m/z): calculated for
C16H21N5O3S [M+H]+ 364.1438; found 364.1444.
4-(4-(Azepan-1-yl)-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (8k)
Yield 272 mg (78 %). White solid, m.p. 199-200 °С. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.11
(d, J= 1.7 Hz, 1H), 7.89 (d, J= 8.4 Hz, 2H), 7.77 (d, J= 8.4 Hz, 2H), 7.42 (s, 2H), 5.71 (d, J= 1.5
Hz, 1H), 3.55 (s, 4H), 1.73 (s, 4H), 1.52 (s, 4H). 13C NMR (101 MHz, DMSO-d6) δ ppm 160.36,
148.52, 144.53, 142.56, 130.74, 126.36, 125.71, 96.37, 49.34, 26.49. HRMS (ESI, m/z):
calculated for C16H20N4O3S [M+H]+ 349.1329; found 349.1328.
4-(4-Morpholino-6-oxopyridazin-1(6H)-yl)benzenesulfonamide (8l)
Yield 299 mg (89 %). White solid, m.p. 193-194°С. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.24
(s, 1H), 7.84 (dd, J= 53.1, 8.4 Hz, 4H), 7.43 (s, 2H), 5.97 (s, 1H), 3.72 (s, 4H), 3.42 (s, 4H). 13C
(101 MHz, DMSO-d6) δ ppm 160.35, 150.14, 144.33, 142.83, 131.59, 126.44, 125.83, 100.29,
65.96, 46.25. HRMS (ESI, m/z): calculated for C14H17N4O4S [M+H]+ 337.0965; found 337.0958.
4.3. Carbonic anhydrase inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
CO2 hydration activity [36]. 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 Tris (pH 8.3) 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 0.005 nM were done thereafter with the
assay buffer. 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 inhibition
constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-
Prusoff equation, as reported earlier, and represent the mean from at least three different
determinations. All CA isoforms were recombinant ones obtained in-house [37-40].
4.4. Cell viability assay
Human cell lines were maintained at 37°C in humidified atmosphere containing air and 5% CO2
as previously described [41]. Retinal pigment epithelial cells ARPE-19 were obtained from

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American Type Culture Collection (ATCC, Manassas, VA, USA). Human melanoma cell line
SK-MEL-2 were obtained from BioloT (Saint Petersburg, Russian Federation). Pancreas ductal
adenocarcinoma cells PANC-1 were obtained from Russian collection cell cultures at the
Institute of Cytology RAS (Saint Petersburg, Russian Federation). Cell line were grown in
Dulbeccos Modified Eagle’s Medium-F12 (BioloT) containing 10% (v/v) heat-inactivated fetal
calf serum (FCS, HyClone Laboratories, UT, USA), 1% L-glutamine, 1% sodium pyruvate, 50
U/mL penicillin, and 50 µg/mL streptomycin (BioloT). Cytotoxicity of carbonic anhydrase
inhibitors was evaluated using a routine colorimetric method with tetrazolium dye – 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The cell lines were incubated for
48 h under normoxia and in the presence of the hypoxia-mimicking agent 50 µM Co2+ with
medium containing different concentrations of carbonic anhydrase inhibitors. Following
treatment, Dulbeccos Modified Eagle’s Medium-F12 (100 µL/ well) and 20 µL of a 2.5 mg/mL
MTT solution were added and cells were incubated for 1 h at 37 °C. The used cell density was 5
× 103 cells/200 µL/well in 96-well microtiter plates. After aspiration of the supernatants, the
MTT-formazan crystals formed by metabolically active cells were dissolved in dimethyl
sulfoxide (100 µL/well) and absorbance was measured at 540 nm and 690 nm in Varioskan
LUX™ Multimode Microplate Reader (Thermo Scientific, USA). Values measured at 540 nm
were subtracted for background correction at 690 nm, and the data were plotted as a percent of
control untreated samples.
4.5. Molecular modeling studies
The crystal structures of hCA II (PDB code 2AW1) and hCA IX (PDB code 3IAI) were taken
from the Protein Data Bank [42]. Molecular docking calculations were performed with
AUTODOCK 4.2 [43] using the improved force field [44]. Autodock Tools were employed for
identifying the torsion angles in the ligand, add the solvent model and assign the Kollman atomic
charges to the protein. Ligand charges were calculated with the Gasteiger method. A grid
spacing of 0.375 Å and a distance-dependent function of the dielectric constant were used for the
energetic map calculations. The ligands were subjected to a robust docking procedure already
used in virtual screening and pose prediction studies [45-46]. Each docked compound was
subjected to 200 runs of the AUTODOCK search using the Lamarckian Genetic Algorithm
performing 10 000 000 steps of energy evaluation. The number of individuals in the initial
population was set to 500 and a maximum of 10 000 000 generations were simulated during each
docking run. All other settings were left as their defaults and the best docked conformations were
taken into account. The selected docking poses were then refined through energy minimization
in explicit water environment [47]. The ligand-protein complexes were minimized employing

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Amber 16 software [48] with ff14SB force field. The complexes were placed in a rectangular
parallelepiped water box, using the TIP3P explicit solvent model for water, and were solvated
with a 15 Å water cap. Sodium ions were added as counter ions to neutralize the system. Two
minimization stages consisting of 5000 steps of steepest descent followed by conjugate gradient,
until a convergence of 0.05 kcal/Å mol, were then performed. In the first one, the protein was
kept rigid with a position restraint of 100 kcal/mol·Å2 to uniquely minimize the positions of the
water molecules. In the second stage, the entire system was energy minimized by applying a
harmonic potential of 10 kcal/mol·Å2 only to the protein α carbons.
Acknowledgements
This research was supported by the Russian Federation Government Megagrant
14.W03.031.0025. We are grateful to the Centre for Chemical Analysis and Materials Research
of Saint Petersburg State University Research Park for the high-resolution mass-spectrometry
data.
A. Supplementary material
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/xxx.
References
[1] C. T. Supuran, Structure and function of carbonic anhydrases. Biochem. J. 473 (2016) 2023–
2032.
[2] V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, D. 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.
[3] C.T. Supuran, Carbonic anhydrase inhibitors and activators for novel therapeutic
applications, Future Med. Chem. 3 (2011) 1165-1180.
[4] C.T. Supuran, C. Capasso, The η-class carbonic anhydrases as drug targets for antimalarial
agents, Expert Opin. Ther. Targets. 19 (2015) 551–563.
[5] C.T. Supuran, Diuretics: from classical carbonic anhydrase inhibitors to novel applications of
the sulfonamides, Curr. Pharm. Des. 14 (2008) 641−648.
[6] C.T. Supuran, A. Scozzafava, F. Mincione, The development of topically acting carbonic
anhydrase inhibitors as antiglaucoma agents, Curr. Pharm. Des. 14 (2008) 649−654.
[7] C.T. Supuran, Carbonic anhydrases: novel therapeutic applications for inhibitors and
activators, Nat. Rev. Drug Discov., 7 (2008) 168-181.
[8] M. Krasavin, M. Korsakov, Z. Zvonaryova, E. Semyonychev, T. Tuccinardi, S. Kalinin,
M. Tanç, C.T. Supuran, Human carbonic anhydrase inhibitory profile of mono- and bis-

ACCEPTED MANUSCRIPT
sulfonamides synthesized via a direct sulfochlorination of 3-and 4-(hetero)arylisoxazol-5-amine
scaffolds, Bioorg. Med. Chem. 25 (2017) 1914-1925.
[9] C.T. Supuran, S. Kalinin, M. Tanç, P. Sarnpitak, P. Mujumdar, S.-A. Poulsen, M. Krasavin,
Isoform-selective inhibitory profile of 2-imidazoline-substituted benzenesulfonamides against a
panel of human carbonic anhydrases, J. Enzyme Inhib. Med. Chem., 31 (Suppl. 1) (2016) 197-
202.
[10] G. La Regina, A. Coluccia, V. Famiglini, S. Pelliccia, L. Monti, D. Vullo, E. Nuti, V.
Alterio, G. De Simone, S. M. Monti, P. Pan, S. Parkkila, C.T. Supuran, A. Rossello, R. Silvestri,
Discovery of 1,1′-Biphenyl-4-sulfonamides as a New Class of Potent and Selective Carbonic
Anhydrase XIV Inhibitors, J. Med. Chem. 58 (2015) 8564-8572.
[11] J. Ivanova, F. Carta, D. Vullo, J. Leitans, A. Kazaks, K. Tars, R. Žalubovskis, C.T. Supuran,
N-Substituted and ring opened saccharin derivatives selectively inhibit transmembrane, tumor-
associated carbonic anhydrases IX and XII, Bioorg. Med. Chem. 25 (2017) 3583-3589.
[12] F. Carta, D. Vullo, S. M. Osman, Z. AlOthman, C. T. Supuran, Synthesis and Carbonic
Anhydrase inhibition of a series of SLC-0111 analogs. Bioorg. Med. Chem. 25 (2017) 2569–
2576.
[13] S. Singh, C. L. Lomelino, M. Y. Mboge, S. C. Frost, R. McKenna, Cancer Drug
Development of Carbonic Anhydrase Inhibitors beyond the Active Site, Molecules 2018 (23)
1045.
[14] L. Schwartz, C. T. Supuran, K. O. Alfarouk, The Warburg effect and the hallmarks of
cancer. Anticancer Agents Med. Chem. 17 (2017) 164–170.
[15] C. T. Supuran, Carbonic Anhydrase Inhibition and the Management of Hypoxic Tumors,
Metabolites 48 (2017) 48.
[16] T. Yamaoka, M. Ohba, T. Ohmori, Molecular-Targeted Therapies for Epidermal Growth
Factor Receptor and Its Resistance Mechanisms. Int. J. Mol. Sci. 18 (2017) E2420.
[17] A. Nocentini, C. T. Supuran, Carbonic anhydrase inhibitors as antitumor/antimetastatic
agents: a patent review (2008-2018). Expert Opin. Ther. Pat. 28 (2018) 729-740.
[18] C.T. Supuran, How many carbonic anhydrase inhibition mechanisms exist?, J. Enzyme
Inhib. Med. Chem. 31 (2016) 345–360.
[19] P.A. Boriack-Sjodin, S. Zeitlin, H.-H. Chen, L. Crenshaw, S. Gross, A. Dantanarayana,
P. Delgado, J.A. May, T. Dean, D.W. Christianson, Structural analysis of inhibitor binding to
human carbonic anhydrase II, Protein Sci. 7 (1998) 2483-2489.
[20] S. Kalinin, S. Kopylov, T. Tuccinardi, A. Sapegin, D. Dar'in, A. Angeli, C. T. Supuran, M.
Krasavin, Lucky Switcheroo: Dramatic Potency and Selectivity Improvement of Imidazoline
Inhibitors of Human Carbonic Anhydrase VII. ACS Med. Chem. Lett. 8 (2017) 1105-1109.
[21] M. Krasavin, M. Korsakov, M. Dorogov, T. Tuccinardi, N. Dedeoglu, C.T. Supuran,
Probing the ‘bipolar nature of the carbonic anhydrase active site: Aromatic sulfonamides
containing 1,3-oxazol-5-yl moiety as picomolar inhibitors of cytosolic CA I and CA II isoforms,
Eur. J. Med. Chem. 101 (2015) 334-347.
[22] M. Krasavin, M. Korsakov, O. Ronzhina, T. Tuccinardi, S. Kalinin, M. Tanç, C. T.
Supuran, Primary mono- and bis-sulfonamides obtained via regiospecific sulfochlorination of N-

ACCEPTED MANUSCRIPT
arylpyrazoles: inhibition profile against a panel of human carbonic anhydrases. J. Enz. Inhib.
Med. Chem. 32 (2017) 920-934.
[23] M. Krasavin, A. Shetnev, T. Sharonova, S. Baykov, T. Tuccinardi, K. Kalinin, A. Angeli, C.
T. Supuran, Heterocyclic Periphery in the Design of Carbonic Anhydrase Inhibitors: 1,2,4-
Oxadiazol-5-yl Benzenesulfonamides as Potent and Selective Inhibitors of Cytosolic hCA II and
Membrane-Bound hCA IX Isoforms. Bioorg. Chem. 76 (2018) 88-97.
[24] R. Yaseen, D. Ekinci, M. Senturk, A. Dh. Hameed, S. Ovais, P. Rathore, M. Samim, K.
Javed, C. T. Supuran, Pyridazinone substituted benzenesulfonamides as potent carbonic
anhydrase inhibitors. Bioorg. Med. Chem. Lett. 26 (2016) 1337-1341.
[25] W. J. Coates, A. McKillop, One-Pot Preparation of 6-Substituted 3(2H)-Pyridazinones from
Ketones. Synthesis (1993) 334-342.
[26] R. Bashir, S. Yaseen, S. Ovais, S. Ahmad, H. Hamid, M.S. Alam, M. Samim, K. Javed,
Synthesis and biological evaluation of some novel sulfamoylphenyl-pyridazinone as anti-
inflammatory agents (Part-II). J. Enzyme Inhib. Med. Chem. 27 (2012) 92-96.
[27] D. J. Katz, D. S. Wise, L. B. Townsend, Synthesis of pyridazine analogs of the naturally
occurring nucleosides cytidine, uridine, deoxycytidine and deoxyuridine. J. Med. Chem. 25
(1982) 813–821.
[28] A. Purohit, H. Radeke, M. Azure, K. Hanson, T. Benetti, F. Su, P. Yalamanchili, M. Yu, M.
Hayes, M. Guaraldi, M. Kagan, S. Robinson, D. Casebier, Synthesis and Biological Evaluation
of Pyridazinone Analogues as Potential Cardiac Positron Emission Tomography Tracers. J. Med.
Chem. 51 (2008) 2954–2970.
[29] P. C. Ting, R. Kuang, H. Wu, R. G. Aslanian, J. Cao, D. W. Kim, J. F. Lee, J. Schwerdt, G.
Zhou, S. Wainhaus, T. A. Black, A. Cacciapuoti, P. M. McNicholas, Y. Xu, S. S. Walker, The
synthesis and structure–activity relationship of pyridazinones as glucan synthase inhibitors.
Bioorg. Med. Chem. Lett. 21 (2011) 1819-1822.
[30] A. Mateus, A. Treyer, C. Wegler, M. Karlgren, P. Matsson, P. Artursson, Intracellular drug
bioavailability: a new predictor of system dependent drug disposition. Sci Rep. 7 (2017) 43047.
[31] A. Grandane, M. Tanc, L. Di Cesare Mannelli, F. Carta, C. Ghelardini, R. Žalubovskis, C.
T. Supuran, Substituted sulfocoumarins are selective carbonic anhdydrase IX and XII inhibitors
with significant cytotoxicity against colorectal cancer cells. J. Med. Chem. 58 (2015) 3975-3983.
[32] K. C. Dunn, A. E. Aotaki-Keen, F. R. Putkey, L. M. Hjelmeland, ARPE-19, a human retinal
pigment epithelial cell line with differentiated properties. Exp. Eye Res. 62 (1996) 155-169.
[33] G. Rui, H. C. Silva, L. Carvalho, M. F. Botelho, A. Mota-Pinto, PaCa-2 and PANC-1 –
pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin
receptors. Sci. Rep. 6 (2016) 21648.
[34] M. S. Al Okail, Cobalt chloride, a chemical inducer of hypoxia-inducible factor-1a in U251
human glioblastoma cell line. J. Saudi Chem. Soc. 14 (2010) 197-201.
[35] A. Caunii, C. Oprean, M. Cristea, A. Ivan, C. Danciu, C. Tatu, V. Paunescu, D. Marti, G.
Tzanakakis, D. A. Spandidos, A. Tsatsakis, R. Susan, C. Soica, S. Avram, C. Dehelean, Effects
of ursolic and oleanolic on SK-MEL-2 melanoma cells: In vitro and in vivo assays. Int. J. Oncol.
51 (2017) 1651–1660.

ACCEPTED MANUSCRIPT
[36] 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.
[37] A. Maresca, F. Carta, D. Vullo, C.T. Supuran, Dithiocarbamates strongly inhibit the β-class
carbonic anhydrases from Mycobacterium tuberculosis, J. Enzyme Inhib. Med. Chem. 28 (2013)
407–411.
[38] D. Ekinci, N.I. Kurbanoglu, E. Salamci, M. Senturk, C.T. Supuran, Carbonic anhydrase
inhibitors: inhibition of human and bovine isoenzymes by benzenesulphonamides, cyclitols and
phenolic compounds, J. Enzyme Inhib. Med. Chem. 27 (2012) 845–848.
[39] D. Ekinci, L. Karagoz, D. Ekinci, M. Senturk, C.T. Supuran, Carbonic anhydrase inhibitors:
in vitro inhibition of α isoforms (hCA I, hCA II, bCA III, hCA IV) by flavonoids, J. Enzyme
Inhib. Med. Chem. 28 (2013) 283–288.
[40] C. Alp, A. Maresca, N.A. Alp, M.S. Gültekin, D. Ekinci, A. Scozzafava, C.T. Supuran,
Secondary/tertiary benzenesulfonamides with inhibitory action against the cytosolic human
carbonic anhydrase isoforms I and II, J. Enzyme Inhib. Med. Chem. 28 (2013) 294–298.
[41] A.V. Hubina, A.A. Pogodaev, V.V. Sharoyko, E.G. Vlakh, T.B. Tennikova, Self-assembled
spin-labeled nanoparticles based on poly(amino acids). React. Funct. Polym. 100 (2016) 173-
180.
[42] H.M. Berman, T. Battistuz, T.N. Bhat, W.F. Bluhm, P.E. Bourne, K. Burkhardt, Z. Feng,
G.L. Gilliland, L. Iype, S. Jain, P. Fagan, J. Marvin, D. Padilla, V. Ravichandran, B. Schneider,
N. Thanki, H. Weissig, J.D. Westbrook, C. Zardecki, The protein data bank, Acta Crystallogr. D:
Biol. Crystallogr. 58 (2002) 899–907.
[43] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson,
AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility, J.
Comput. Chem. 30 (2009) 2785–2791.
[44] D. Santos-Martins, S. Forli, M.J. Ramos, A.J. Olson, AutoDock4(Zn): an improved
AutoDock force field for small-molecule docking to zinc metalloproteins, J. Chem. Inf. Model.
54 (2014) 2371–2379.
[45] G. Poli, A. Gelain, F. Porta, A. Asai, A. Martinelli, T. Tuccinardi T. Identification of a new
STAT3 dimerization inhibitor through a pharmacophore-based virtual screening approach. J.
Enzyme. Inhib. Med. Chem. 31 (2016) 1011-1017.
[46] L. Milella, S. Milazzo, M. De Leo, M.B. Vera Saltos, I. Faraone, T. Tuccinardi, M. Lapillo,
N. De Tommasi, A. Braca. α-Glucosidase and α-Amylase Inhibitors from Arcytophyllum
thymifolium. J. Nat. Prod. 79 (2016) 2104-2112.
[47] M. Aghazadeh Tabrizi, P.G. Baraldi, E. Ruggiero, G. Saponaro, S. Baraldi, G. Poli, T.
Tuccinardi, A. Ravani, F. Vincenzi, P.A. Borea, K. Varani. Synthesis and structure activity
relationship investigation of triazolo[1,5-a]pyrimidines as CB2 cannabinoid receptor inverse
agonists. Eur. J. Med. Chem. 113 (2016) 11-27.
[48] D.A. Case, J.T. Berryman, R.M. Betz, D.S. Cerutti, Darden, R.E. Duke, T.J. Giese, H.
Gohlke, A.W. Goetz, N. Homeyer, S. Izadi, P. Janowski, J. Kaus, A. Kovalenko, T.S. Lee, S.
LeGrand, P. Li, T. Luchko, R. Luo, B. Madej, K.M. Merz, G. Monard, P. Needham, H. Nguyen,
H.T. Nguyen, I. Omelyan, A. Onufriev, D.R. Roe, A. Roitberg, R. Salomon-Ferrer, C.L.
Simmerling, W. Smith, J. Swails, R.C. Walker, J. Wang, R.M. Wolf, X. Wu, D.M. York, P.A.
Kollman, AMBER, Version 16, University of California, San Francisco, CA.

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•
•
•
•
•
A small set of pyridazine carbonic anhydrase inhibitors (CAIs) was reported earlier
A larger set was designed which delivered potent inhibitors of CA IX isoform
This membrane-bound isoform is considered a promising cancer target
Two compounds showed potential to selectively kill cancer cells
SAR findings have been rationalized by in silico docking experiments