Inhibition studies on carbonic anhydrase isoforms I, II, IV and IX with N-arylsubstituted secondary sulfonamides featuring a bicyclic tetrahydroindazole scaffold

Article abstract of DOI:10.1016/j.ejmech.2021.113490

Carbonic Anhydrases (CAs) are pharmaceutically relevant targets for the treatment of several disease conditions. The ubiquitous localization of these enzymes and the high homology shared by the different isoforms represent substantial impediments for the

Full text of DOI:10.1016/j.ejmech.2021.113490

European Journal of Medicinal Chemistry 220 (2021) 113490
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Inhibition studies on carbonic anhydrase isoforms I, II, IV and IX with
N-arylsubstituted secondary sulfonamides featuring a bicyclic
tetrahydroindazole scaffold
Silvia Salerno a, 1, Giorgio Amendola b , Andrea Angeli , Emma Baglini ,
,
1
c
a
a
a
b
d
Elisabetta Barresi , Anna Maria Marini , Rahul Ravichandran , Monica Viviano ,
d
e
a
c
Sabrina Castellano , Ettore Novellino , Federico Da Settimo , Claudiu T. Supuran ,
b
,
*
a
Sandro Cosconati , Sabrina Taliani
a
Department of Pharmacy, University of Pisa, Pisa, Italy
b
DiSTABiF, University of Campania Luigi Vanvitelli, Caserta, Italy
NEUROFARBA Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universit aꢀ degli Studi di Firenze, Sesto Fiorentino (Florence), Italy
Department of Pharmacy, Epigenetic Med Chem Lab, University of Salerno, Fisciano (SA), Italy
Department of Pharmacy, University Federico II of Naples, Naples, Italy
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 December 2020
Received in revised form
Carbonic Anhydrases (CAs) are pharmaceutically relevant targets for the treatment of several disease
conditions. The ubiquitous localization of these enzymes and the high homology shared by the different
isoforms represent substantial impediments for the discovery of potential drugs devoid of off-target side
effects. As a consequence, substantial efforts are still needed to allow for the full realization of the
pharmacological potential of CA modulators. In this contribution, starting from our previous studies, we
describe the synthesis of a set of new bicyclic tetrahydroindazoles featuring a secondary sulfonamide.
Biological evaluation of the inhibitory activity against the hCA I, II, IV, and IX isoforms allowed drawing a
structure-activity relationship profile that was rationalized through theoretical studies. This allowed
dissecting the new molecules into the single portions influencing the zinc chelation properties and the
selectivity profile thereby offering a new platform for the discovery of new isotype selective CA
inhibitors.
24 March 2021
Accepted 17 April 2021
Available online 24 April 2021
Keywords:
Secondary sulfonamides
Carbonic anhydrase inhibitors
Structure-activity relationships
©
2021 Elsevier Masson SAS. All rights reserved.
1
. Introduction
To date, fifteen CA isoforms have been identified in humans, that
differ in the catalytic activity, tissue distribution, and subcellular
localization: the cytosolic CA IeIII, VII and XIII; the mitochondrial
CA VA and VB; the secreted CA VI; the membrane-bound CA IV, IX,
XII, and XIV [1].
CA inhibition constitutes an appealing target for medicinal
chemists interested in the development of novel small molecules
potentially useful as diuretics and for the treatment of several
conditions including glaucoma, epilepsy, obesity, and cancer [5e8].
However, the high sequence homology across CA isoforms in
humans, combined with their diffuse and ubiquitous tissue locali-
zation, makes the discovery of CAIs endowed with functional
selectivity for a specific target isoform one of the primary chal-
lenges in the development of potential drugs devoid of off-target
side effects [9].
Primary sulfonamides represent the classical inhibitors of car-
bonic anhydrases (CAs), ubiquitary metalloenzymes that catalyze
the simple two-steps reversible hydration of carbon dioxide to bi-
carbonate and play a role in crucial physiological processes, such as
regulation of intracellular pH and cell homeostasis [1e3]. CA in-
hibitors (CAIs) coordinate the zinc ion present in the catalytic site
through their deprotonated sulfonamide nitrogen atom and a
number of them (acetazolamide, ethoxzolamide, dichlorphena-
mide) have been in clinical use for many decades as diuretics and
systemically acting antiglaucoma drugs [4].
*
Corresponding author.
E-mail address: sandro.cosconati@unicampania.it (S. Cosconati).
Equal contribution.
More recently, efforts were directed towards secondary and
1
https://doi.org/10.1016/j.ejmech.2021.113490
223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
0

S. Salerno, G. Amendola, A. Angeli et al.
European Journal of Medicinal Chemistry 220 (2021) 113490
tertiary sulfonamides as an alternative strategy to identify CAIs
with promising activity and selectivity profiles; in some cases, the
mechanism of action of such compounds was hypothesized as be-
ing different from that of the primary analogs and not fully clarified
Table 1
Inhibition of hCA isoforms I, II, IV, and IX with indazolyl-N-arylbenzenesulfonamides
e27 and AAZ as the reference standard by a stopped-flow CO hydrase assay.
1
2
[
10e13].
In the last decade, different tricyclic/bicyclic chemotypes have
been exploited by our research group in search of novel CAIs,
including benzo- or pyrido-thiopyranopyrazole I, benzo- or pyrido-
thiopyranopyrimidine II, 4,5-dihydro-pyrazoloquinazoline III, tet-
rahydroquinazole IV, and tetrahydroindazole V (Chart 1) [14e16].
All these scaffolds were decorated with a primary sulfonamide
moiety. Our efforts allowed for the identification of a number of
derivatives possessing high inhibitory activity and variable selec-
tivity profiles [14e16].
Specifically, compounds from class I behaved in general as in-
hibitors of human (h) CA I and II isoforms [14], whereas an excellent
inhibitory activity against hCA II, IX, and XII was shown by most of
sulfonamides II [15]. Many highly effective and selective inhibitors
against hCA IX were identified within classes III-V [16].
cpd
R1
H
R2
R3
Ki (m
M)a
hCA I
hCA II
hCA IV
hCA IX
V a
V b
V c
1
2
3
4
5
6
7
H
H
CH
H
H
H
H
H
H
H
H
H
-
-
-
H
0.746
0.296
0.272
5.7
1.4
6.3
5.7
4.9
82.5
5.3
5.2
0.078
0.0052
0.0086
31.0
5.9
27.0
50.3
8.4
>100
21.2
>100
35.1
23.6
0.41
2.1
5.8
7.4
4.4
8.8
6.5
7.6
26.5
2.6
3.5
0.0095
0.214
0.040
59.6
79.3
68.3
75.8
52.5
>100
55.4
71.0
56.9
83.2
62.9
73.5
0.93
78.2
87.2
65.0
>100
>100
82.1
49.7
56.3
74.7
58.0
47.8
55.7
92.6
79.0
0.074
0.0052
0.00073
0.00088
97.9
56.5
>100
83.7
39.2
>100
37.1
>100
33.0
95.4
40.0
47.0
4.1
46.7
57.3
41.8
84.7
61.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.026
6
C H
5
CH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3
3
p-OCH
p-Cl
p-NO
p-CH
m-OCH
m-Cl
m-NO
m-CH
H
p-OCH
p-Cl
p-NO
p-CH
m-OCH
m-Cl
m-NO
m-CH
H
p-OCH
p-Cl
p-NO
p-CH3
m-OCH
m-Cl
m-NO
m-CH
3
In the present study, we aimed at expanding the chemical di-
versity of the so far rarely explored secondary sulfonamide CAIs by
functionalizing our previously described tetrahydroindazoles. Here
the idea was to pursue new design strategies different from the
classical tail approach that is normally followed in the discovery of
new CAIs. For this purpose, derivatives 1e27 (Table 1) were syn-
thesized and biologically evaluated for their enzyme inhibitory
capacity against four physiologically relevant CA isoforms, the hCA
I, II, IV, and IX. In the novel compounds the secondary benzene-
sulfonamide moiety was decorated at para- or meta-position with
groups characterized by different electron-donorꢀacceptor capa-
2
3
3
8
9
2
3
5.5
45.9
8.6
1
1
0
1
C
C
6
H
H
5
6
5
3
12
13
C6H5
32.2
52.2
60.3
40.3
69.1
57.9
48.8
82.3
64.4
9.1
>100
>100
15.0
>100
>100
>100
0.25
C
6
C
6
C
6
C
6
C
6
C
6
H
H
H
H
H
H
5
5
5
5
5
5
2
14
15
16
17
3
3
bilities (R
3
¼ H, OCH
3 2 3
, Cl, NO , CH ) to modulate the acidity of the
H
H
CH
CH
CH
CH
2
sulfonamide itself, while the same substituents as the corre-
sponding primary sulfonamides recently described by us [16] were
18
3
19
20
21
22
3
3
3
3
CH
CH
CH
CH
3
3
3
3
3
maintained at 6-position (R
1
¼ R
2
¼ H; R
1
¼ R
2
¼ CH
3
; R
1
¼ H,
R
2
¼ C ). Computational studies were performed to predict the
6 5
H
2
34.8
4.0
4.1
>100
57.9
>100
0.012
mode of interactions and provide a rationalization of the acquired
structure-activity relationship (SAR) data to prospectively guide the
design of more active and isoform-selective CAIs.
23
24
CH3
CH3
CH
CH
CH
CH
3
3
3
3
CH
CH
CH
CH
3
3
3
3
3
25
26
27
2
3
2
. Experimental section
AAZ
a
Mean from three different assays. Errors were in the range of ±5e10% of the
2.1. Chemistry
reported values (data not shown).
The uncorrected melting points were determined using a
Chart 1. Structures of previously described (I-V) and newly synthesized (1-27) CAIs.
2

S. Salerno, G. Amendola, A. Angeli et al.
European Journal of Medicinal Chemistry 220 (2021) 113490
Reichert K o€ fler hot-stage apparatus. NMR spectra were obtained on
150.5 (CeAr), 156.8 (CeAr), 192.6 (C]O).
1
13
a Bruker AVANCE 400 ( H, 400 MHz, C, 100 MHz) in DMSO‑d
6
.
Chemical shifts are expressed in (ppm) and coupling constants (J)
d
2
1
.1.1.3. N-(4-Chlorophenyl)-4-(4-oxo-4,5,6,7-tetrahydro-1H-indazol-
-yl)benzenesulfonamide (3). Compound 3 was obtained as a solid
in Hertz. Magnesium sulfate was used as the drying agent. Evapo-
rations were made in vacuo (rotating evaporator). Analytical TLC
has been carried out on Merck 0.2 mm precoated silica gel
aluminum sheets (60 F-254). Silica gel 60 (230e400 mesh) was
used for column chromatography. The purity of the target in-
hibitors 1e27 was determined, using a Shimadzu LC-20AD SP liquid
chromatograph equipped with a DDA Detector at 220 nm (column
(94 mg, 25%) starting from compound 30a (0.27 g) and 1-chloro-4-
ꢂ
1
iodobenzene (0.27 g); m.p.: 159e162 C; H NMR (400 MHz,
DMSO‑d ):
2.04e2.07 (m, 2H), 2.44 (t, J ¼ 6.4 Hz, 2H), 3.05 (t,
J ¼ 6.0 Hz, 2H), 7.13 (AA'XX’, JAA’/XX’ ¼ 2.6 Hz, JAX ¼ 9.2 Hz, 2H), 7.33
AA'XX’, JAA’/XX’ ¼ 2.4 Hz, JAX ¼ 8.8 Hz, 2H), 7.84 (AA'XX’, JAA’/
6
d
(
XX’ ¼ 2.2 Hz, JAX ¼ 8.8 Hz, 2H), 7.91 (AA'XX’, JAA’/XX’ ¼ 2.4 Hz,
C18 (250 mm ꢁ 4.6 mm, 5
delivered at isocratic flow, consisted of methanol (80%) and water
20%) and a flow rate of 1.0 mL/min. All the compounds showed
mm, Shim-pack)). The mobile phase,
13
J
AX ¼ 9.2 Hz, 2H), 8.10 (s, 1H), 10.58 (s, 1H). C NMR (100 MHz,
6
DMSO‑d ): d 22.8 (C-6), 23.1 (C-7), 37.4 (C-5), 120.7 (C-3a), 121.9
(
(
(
(
2CeAr), 123.6 (2CeAr), 128.2 (2CeAr), 128.6 (CeAr), 129.3
2CeAr), 136.5 (CeAr), 138.1 (C-7a), 138.7 (CeAr), 141.8 (C-3), 150.6
CeAr), 192.7 (C]O).
percent purity values of >95%. Reagents, starting materials, and
solvents were purchased from commercial suppliers and used as
received.
The following intermediates were obtained according to previ-
ously described procedures: 2-[(dimethylamino)methylene]cyclo-
hexan-1,3-dione 29a [17], 2-[(dimethylamino)methylene]-5-
phenylcyclohexan-1,3-dione 29b [17], 2-[(dimethylamino)methy-
lene]-5,5-dimethylcyclohexan-1,3-dione 29c [17], 4-(4-oxo-4,5,6,7-
tetrahydro-1H-indazol-1-yl)benzenesulfonamide 30a [16], 4-(4-
oxo-6-phenyl-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzenesulfona-
mide 30b [16], 4-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-inda-
zol-1-yl)benzenesulfonamide 30c [16].
2
1
.1.1.4. N-(4-Nitrophenyl)-4-(4-oxo-4,5,6,7-tetrahydro-1H-indazol-
-yl)benzenesulfonamide (4). Compound 4 was obtained as a solid
(77 mg, 20%) starting from compound 30a (0.27 g) and 1-iodo-4-
ꢂ
1
nitrobenzene (0.28 g); m.p.: 215e217 C; H NMR (400 MHz,
DMSO‑d ):
2.03e2.06 (m, 2H), 2.44 (t, J ¼ 6.2 Hz, 2H), 3.05 (t,
J ¼ 6.2 Hz, 2H), 7.35 (d, J ¼ 9.2 Hz, 2H), 7.87 (d, J ¼ 8.8 Hz, 2H), 8.05
d, J ¼ 8.8 Hz, 2H), 8.10 (s, 1H), 8.16 (d, J ¼ 9.2 Hz, 2H), 11.43 (s, 1H).
6
d
(
13
6
C NMR (100 MHz, DMSO‑d ): d 22.9 (C-6), 23.1 (C-7), 37.5 (C-5),
118.3 (2CeAr), 120.8 (C-3a), 123.9 (2CeAr), 125.6 (2CeAr), 128.4
(
(
2CeAr), 137.9 (C-7a), 138.9 (CeAr), 142.2 (C-3), 142.9 (CeAr), 144.0
CeAr), 150.8 (CeAr), 192.8 (C]O).
2
4
.1.1. General procedure for the synthesis of N-aryl-4-(4-oxo-
,5,6,7-tetrahydro-1H-indazol-1-yl)benzenesulfonamide derivatives
(
1e27)
To a solution of the proper 4-(4-oxo-6-substituted-4,5,6,7-
tetrahydro-1H-indazol-1-yl)benzenesulfonamide 30a-c
0.94 mmol) in dry MeCN (10.0 mL) at room temperature, CuI
8 mg, 0.04 mmol), anhydrous K CO (0.28 g, 2.07 mmol), the
2
.1.1.5. 4-(4-Oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)-N-(p-tolyl)
benzenesulfonamide (5). Compound 5 was obtained as a solid
72 mg, 20%) starting from compound 30a (0.27 g) and 1-iodo-4-
(
(
(
ꢂ
1
methylbenzene (0.25 g); m.p.: 128e130 C; H NMR (400 MHz,
DMSO‑d ):
2.04e2.07 (m, 2H), 2.19 (s, 3H), 2.44 (t, J ¼ 6.2 Hz, 2H),
3.05 (t, J ¼ 6.0 Hz, 2H), 7.00 (d, J ¼ 8.4 Hz, 2H), 7.05 (d, J ¼ 8.0 Hz,
2
3
6
d
proper aryl iodide (1.13 mmol) and DMEDA (0.04 mL, 0.38 mmol)
were added, and the resulting mixture was heated at 100 C for
8
ꢂ
2
H), 7.82 (d, J ¼ 8.4 Hz, 2H), 7.88 (d, J ¼ 8.8 Hz, 2H), 8.10 (s,1H),10.25
e24 h (TLC analysis). After cooling, the reaction mixture was
13
(
(
(
6 3
s, 1H). C NMR (100 MHz, DMSO‑d ): d 20.4 (CH ), 22.8 (C-6), 23.1
C-7), 37.4 (C-5), 120.6 (C-3a), 121.0 (2CeAr), 123.5 (2CeAr), 128.2
2CeAr), 129.7 (2CeAr), 133.8 (CeAr), 134.8 (CeAr), 138.5 (C-7a),
diluted with water (20 mL), acidified with 2.0 N HCl to pH ¼ 4, and
extracted with EtOAc (3 ꢁ 20 mL). The organic phase was dried
(
4
MgSO ), filtered and evaporated under reduced pressure. The
138.7 (CeAr), 141.5 (C-3), 150.6 (CeAr), 192.7 (C]O).
crude material was purified by flash chromatography using PE/
EtOAc as eluting system, to afford compounds 1e27.
2
.1.1.6. N-(3-Methoxyphenyl)-4-(4-oxo-4,5,6,7-tetrahydro-1H-inda-
zol-1-yl)benzenesulfonamide (6). Compound 6 was obtained as a
solid (0.11 g, 30%) starting from compound 30a (0.27 g) and 3-
2
.1.1.1. 4-(4-Oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)-N-phenyl-
benzenesulfonamide (1). Compound 1 was obtained as a solid
86 mg, 25%) starting from compound 30a (0.27 g) and iodo-
ꢂ
1
iodoanisole (0.13 mL); m.p.: 102e104 C; H NMR (400 MHz,
DMSO‑d ):
2.04e2.07 (m, 2H), 2.44 (t, J ¼ 6.2 Hz, 2H), 3.05 (t,
J ¼ 5.8 Hz, 2H), 3.67 (s, 3H), 6.61e6.64 (m, 1H), 6.70e6.72 (m, 2H),
.15 (t, J ¼ 8.2 Hz,1H), 7.84 (d, J ¼ 8.4 Hz, 2H), 7.94 (d, J ¼ 8.8 Hz, 2H),
(
ꢂ
1
d
benzene (0.13 mL); m.p.128e130 C; H NMR (400 MHz, DMSO‑d
d
6
):
6
2.04e2.08 (m, 2H), 2.44 (t, J ¼ 6.0 Hz, 2H), 3.04 (t, J ¼ 6.0 Hz, 2H),
7
7
.03 (t, J ¼ 7.2 Hz, 1H), 7.11e7.13 (m, 2H), 7.23e7.27 (m, 2H), 7.83 (d,
13
13
8.09 (s, 1H), 10.43 (s, 1H). C NMR (100 MHz, DMSO‑d ):
d
22.8 (C-
), 106.0 (CeAr), 109.4 (CeAr),
12.1 (CeAr), 120.6 (C-3a), 123.5 (2CeAr), 128.2 (2CeAr), 130.2
J ¼ 9.2 Hz, 2H), 7.92 (d, J ¼ 8.8 Hz, 2H), 8.10 (s, 1H), 10.42 (s, 1H).
C
6
6), 23.1 (C-7), 37.4 (C-5), 55.1 (OCH
3
6
NMR (100 MHz, DMSO‑d ): d 22.8 (C-6), 23.1 (C-7), 37.3 (C-5), 120.4
1
(
1
1
C-3a), 120.6 (2CeAr), 123.5 (2CeAr), 124.4 (CeAr), 128.2 (2CeAr),
29.3 (2CeAr), 137.5 (C-7a), 138.7 (CeAr), 141.7 (C-3), 150.6 (CeAr),
79.6 (CeAr), 192.7 (C]O).
(
(
CeAr), 138.4 (CeAr), 138.7 (C-7a), 138.7 (CeAr), 141.6 (C-3), 150.6
CeAr), 159.8 (CeAr), 192.7 (C]O).
2
.1.1.2. N-(4-Methoxyphenyl)-4-(4-oxo-4,5,6,7-tetrahydro-1H-inda-
2.1.1.7. N-(3-Chlorophenyl)-4-(4-oxo-4,5,6,7-tetrahydro-1H-indazol-
1-yl)benzenesulfonamide (7). Compound 7 was obtained as a solid
(0.11 g, 30%) starting from compound 30a (0.27 g) and 3-
zol-1-yl)benzenesulfonamide (2). Compound 2 was obtained as a
solid (75 mg, 20%) starting from compound 30a (0.27 g) and 4-
ꢂ
1
ꢂ
1
iodoanisole (0.26 g); m.p.: 162e164 C; H NMR (400 MHz,
DMSO‑d ):
2.05e2.08 (m, 2H), 2.45 (t, J ¼ 6.4 Hz, 2H), 3.05 (t,
J ¼ 6.0 Hz, 2H), 3.67 (s, 3H), 6.82 (AA'XX’, JAA’/XX’ ¼ 2.0 Hz,
AX ¼ 8.8 Hz, 2H), 7.00 (AA'XX’, JAA’/XX’ ¼ 2.4 Hz, JAX ¼ 9.2 Hz, 2H),
chloroiodobenzene (0.14 mL); m.p.: 124e126 C;
(400 MHz, DMSO‑d ):
H NMR
6
d
6
d
2.02e2.08 (m, 2H), 2.44 (t, J ¼ 6.4 Hz, 2H),
3.05 (t, J ¼ 6.2 Hz, 2H), 7.09e7.16 (m, 3H), 7.29 (t, J ¼ 8.0 Hz,1H), 7.85
J
(d, J ¼ 8.8 Hz, 2H), 7.95 (d, J ¼ 8.8 Hz, 2H), 8.11 (s, 1H), 10.74 (s, 1H).
13
13
7
(
(
.80e7.85 (m, 4H), 8.10 (s, 1H), 10.04 (s, 1H) ppm. C NMR
100 MHz, DMSO‑d ): 22.8 (C-6), 23.0 (C-7), 37.4 (C-5), 55.2
OCH ), 114.4 (2CeAr), 120.6 (C-3a), 123.4 (2CeAr), 123.8 (2CeAr),
28.2 (2CeAr), 129.8 (CeAr), 138.5 (C-7a), 138.6 (CeAr), 141.4 (C-3),
6
C NMR (100 MHz, DMSO‑d ): d 22.9 (C-6), 23.1 (C-7), 37.4 (C-5),
6
d
118.3 (CeAr), 119.4 (CeAr), 120.7 (C-3a), 123.7 (2CeAr), 124.1
(CeAr), 128.3 (2CeAr), 131.1 (CeAr), 133.5 (CeAr), 138.0 (CeAr),
138.8 (C-7a), 139.1 (CeAr), 141.9 (C-3), 150.6 (CeAr), 192.7 (C]O).
3
1
3

S. Salerno, G. Amendola, A. Angeli et al.
European Journal of Medicinal Chemistry 220 (2021) 113490
2
1
.1.1.8. N-(3-Nitrophenyl)-4-(4-oxo-4,5,6,7-tetrahydro-1H-indazol-
-yl)benzenesulfonamide (8). Compound 8 was obtained as a solid
2.1.1.13. N-(4-Nitrophenyl)-4-(4-oxo-6-phenyl-4,5,6,7-tetrahydro-
1H-indazol-1-yl)benzenesulfonamide (13). Compound 13 was ob-
tained as a solid (0.37 g, 80%) starting from compound 30b (0.34 g)
(
97 mg, 25%) starting from compound 30a (0.27 g) and 1-iodo-3-
ꢂ
1
ꢂ
1
nitrobenzene (0.28 g); m.p.: 185e187 C; H NMR (400 MHz,
DMSO‑d ):
2.03e2.07 (m, 2H), 2.44 (t, J ¼ 6.2 Hz, 2H), 3.05 (t,
and 1-iodo-4-nitrobenzene (0.28 g); m.p.: 137e139 C; H NMR
(400 MHz, DMSO‑d ): 2.54e2.55 (m, 1H), 2.94e3.01 (m, 1H),
6
d
6
d
J ¼ 6.0 Hz, 2H), 7.57e7.58 (m, 2H), 7.86 (d, J ¼ 8.4 Hz, 2H), 7.89e7.92
3.14e3.19 (m, 1H), 3.41e3.52 (m, 2H), 7.24e7.28 (m, 1H), 7.32e7.40
(m, 6H), 7.89 (AA'XX’, JAA’/XX’ ¼ 2.2 Hz, JAX ¼ 9.2 Hz, 2H), 8.03
(AA'XX’, JAA’/XX’ ¼ 2.0 Hz, JAX ¼ 8. 8 Hz, 2H), 8.14 (AA'XX’, JAA’/
13
(
(
(
1
(
m, 2H), 7.98 (d, J ¼ 8.8 Hz, 2H), 8.09 (s, 1H), 11.08 (s, 1H). C NMR
100 MHz, DMSO‑d ): 22.8 (C-6), 23.0 (C-7), 37.4 (C-5), 113.7
CeAr), 118.8 (CeAr), 120.7 (C-3a), 123.7 (2CeAr), 125.6 (CeAr),
28.2 (2CeAr), 130.9 (CeAr), 137.8 (CeAr), 138.7 (C-7a), 138.9
CeAr), 142.0 (C-3), 148.3 (CeAr), 150.6 (CeAr), 192.6 (C]O).
6
d
13
XX’ ¼ 2.2 Hz, JAX ¼ 9.2 Hz, 2H), 8.17 (s, 1H), 11.41 (s, 1H). C NMR
(100 MHz, DMSO‑d
(2CeAr), 120.6 (C-3a), 123.7 (2CeAr), 125.4 (2CeAr), 126.8 (CeAr),
27.1 (2CeAr), 128.3 (2CeAr), 128.5 (2CeAr), 137.8 (CeAr), 138.7 (C-
6
): d 30.5 (C-6), 41.1 (C-7), 44.4 (C-5), 118.1
1
2
.1.1.9. 4-(4-Oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)-N-(3-tolyl)
benzenesulfonamide (9). Compound 9 was obtained as a solid
90 mg, 25%) starting from compound 30a (0.27 g) and 3-
7a), 141.9 (C-3), 142.7 (CeAr), 142.9 (CeAr), 143.8 (CeAr), 149.9
(CeAr), 191.4 (C]O).
(
ꢂ
1
iodotoluene (0.14 mL); m.p.: 139e141 C; H NMR (400 MHz,
DMSO‑d ):
2.04e2.07 (m, 2H), 2.20 (s, 3H), 2.44 (t, J ¼ 6.2 Hz, 2H),
.05 (t, J ¼ 6.2 Hz, 2H), 6.86 (d, J ¼ 8.0 Hz, 1H), 6.92e6.94 (m, 2H),
2.1.1.14. 4-(4-Oxo-6-phenyl-4,5,6,7-tetrahydro-1H-indazol-1-yl)-N-
(p-tolyl)benzenesulfonamide (14). Compound 14 was obtained as a
solid (0.13 g, 30%) starting from compound 30b (0.34 g) and 1-iodo-
6
d
3
ꢂ
1
7
.12 (t, J ¼ 7.8 Hz, 1H), 7.83 (d, J ¼ 8.8 Hz, 2H), 7.92 (d, J ¼ 8.8 Hz, 2H),
4-methylbenzene (0.25 g); m.p.: 101e103 C; H NMR (400 MHz,
DMSO‑d ): 2.16 (s, 3H), 2.54e2.55 (m, 1H), 2.93e3.01 (m, 1H),
13
8
.10 (s, 1H), 10.36 (s, 1H). C NMR (100 MHz, DMSO‑d
), 22.8 (C-6), 23.1 (C-7), 37.4 (C-5), 117.3 (CeAr), 120.6 (CeAr),
20.8 (C-3a), 123.5 (2CeAr), 125.1 (CeAr), 128.2 (2CeAr), 129.1
CeAr), 137.4 (CeAr), 138.6 (C-7a), 138.7 (CeAr), 141.6 (C-3), 150.6
CeAr), 192.7 (C]O).
6
):
d
21.1
6
d
(
CH
3
3.10e3.15 (m, 1H), 3.40e3.56 (m, 2H), 6.97 (d, J ¼ 8.4 Hz, 2H), 7.02
(d, J ¼ 8.4 Hz, 2H), 7.24e7.28 (m, 1H), 7.32e7.40 (m, 4H), 7.82
(AA'XX’, JAA’/XX’ ¼ 2.4 Hz, JAX ¼ 8.8 Hz, 2H), 7.86 AA'XX’, JAA’/
1
(
(
13
XX’ ¼ 2.0 Hz, JAX ¼ 8.8 Hz, 2H), 8.16 (s, 1H), 10.21 (s, 1H). C NMR
6 3
(100 MHz, DMSO‑d ): d 20.7 (CH ), 31.0 (C-6), 41.6 (C-7), 44.9 (C-5),
2
.1.1.10. 4-(4-Oxo-6-phenyl-4,5,6,7-tetrahydro-1H-indazol-1-yl)-N-
121.0 (C-3a), 121.4 (2CeAr), 123.9 (2CeAr), 127.4 (CeAr), 127.6
(2CeAr), 128.7 (2CeAr),129.0 (2CeAr),130.1 (2CeAr),134.2 (CeAr),
135.2 (CeAr), 138.4 (C-7a), 139.1 (CeAr), 141.8 (C-3), 143.4 (CeAr),
150.3 (CeAr), 191.9 (C]O).
phenylbenzenesulfonamide (10). Compound 10 was obtained as a
solid (83 mg, 20%) starting from compound 30b (0.34 g) and
ꢂ
1
iodobenzene (0.13 mL); m.p.: 98e100 C; H NMR (400 MHz,
DMSO‑d ): 2.54e2.55 (m, 1H), 2.93e3.01 (m, 1H), 3.11e3.16 (m,
H), 3.40e3.53 (m, 2H), 7.02 (t, J ¼ 7.2 Hz, 1H), 7.10 (d, J ¼ 8.0 Hz,
H), 7.21e7.27 (m, 3H), 7.32e7.40 (m, 4H), 7.83 (d, J ¼ 8.8 Hz, 2H),
6
d
1
2
2.1.1.15. N-(3-Methoxyphenyl)-4-(4-oxo-6-phenyl-4,5,6,7-
tetrahydro-1H-indazol-1-yl)benzenesulfonamide
(15).
13
7
.90 (d, J ¼ 8.8 Hz, 2H), 8.16 (s, 1H), 10.40 (s, 1H). C NMR (100 MHz,
DMSO‑d ): 31.0 (C-6), 41.5 (C-7), 44.9 (C-5), 120.7 (2CeAr), 121.0
C-3a), 123.9 (2CeAr), 124.8 (CeAr), 127.4 (CeAr), 127.6 (2CeAr),
Compound 15 was obtained as a solid (85 mg, 20%) starting from
compound 30b (0.34 g) and 3-iodoanisole (0.13 mL); m.p.:
6
d
ꢂ
1
(
90e92 C; H NMR (400 MHz, DMSO‑d ): d 2.54e2.55 (m, 1H),
6
1
28.7 (2CeAr),129.0 (2CeAr),129.7 (2CeAr),137.9 (CeAr),138.9 (C-
2.94e3.01 (m, 1H), 3.11e3.16 (m, 1H), 3.40e3.53 (m, 2H), 3.64 (s,
3H), 6.59e6.61 (m, 1H), 6.66e6.70 (m, 2H), 7.13 (t, J ¼ 8.0 Hz, 1H),
7.26 (tt, J ¼ 1.6 Hz, J ¼ 7.6 Hz, 1H), 7.32e7.40 (m, 4H), 7.84 (AA'XX’,
7
a), 139.1 (CeAr), 141.9 (C-3), 143.4 (CeAr), 150.3 (CeAr), 191.9 (C]
O).
J
J
AA’/XX’ ¼ 2.0 Hz, JAX ¼ 9.2 Hz, 2H), 7.86 (AA'XX’, JAA’/XX’ ¼ 2.0 Hz,
13
2
.1.1.11. N-(4-methoxyphenyl)-4-(4-oxo-6-phenyl-4,5,6,7-
AX ¼ 9.2 Hz, 2H), 8.17 (s, 1H), 10.22 (s, 1H). C NMR (100 MHz,
): 30.6 (C-6), 41.2 (C-7), 44.5 (C-5), 55.1 (OCH ), 106.0
tetrahydro-1H-indazol-1-yl)benzenesulfonamide
(11).
DMSO‑d
6
d
3
Compound 11 was obtained as a solid (89 mg, 20%) starting from
compound 30b (0.34 g) and 4-iodoanisole (0.26 g); m.p.:
(CeAr), 109.4 (CeAr), 112.2 (CeAr), 120.6 (C-3a), 123.6 (2CeAr),
125.1 (CeAr), 127.0 (CeAr), 127.2 (2CeAr), 128.4 (CeAr), 128.6
(2CeAr), 130.2 (2CeAr), 138.5 (CeAr), 138.7 (C-7a), 141.6 (C-3),
143.1 (CeAr), 150.0 (CeAr), 159.8 (CeAr), 191.6 (C]O).
08e110 C; 1H NMR (400 MHz, DMSO‑d
ꢂ
): d 2.55e2.56 (m, 1H),
1
6
2
3
1
.94e3.01 (m, 1H), 3.11e3.16 (m, 1H), 3.41e3.56 (m, 2H), 3.64 (s,
H), 6.80 (d, J ¼ 9.2 Hz, 2H), 6.98 (d J ¼ 8.8 Hz, 2H), 7.24e7.28 (m,
13
H), 7.32e7.40 (m, 4H), 7.82 (m, 4H), 8.17 (s, 1H), 10.01 (s, 1H).
C
2.1.1.16. N-(3-Chlorophenyl)-4-(4-oxo-6-phenyl-4,5,6,7-tetrahydro-
1H-indazol-1-yl)benzenesulfonamide (16). Compound 16 was ob-
tained as a solid (0.16 g, 35%) starting from compound 30b (0.34 g)
NMR (100 MHz, DMSO‑d
OCH ), 114.4 (2CeAr), 119.5 (CeAr), 120.5 (C-3a), 123.4 (2CeAr),
23.8 (2CeAr), 127.1 (2CeAr), 128.3 (2CeAr), 128.6 (2CeAr), 138.5
6
): d 30.5 (C-6), 41.2 (C-7), 44.5 (C-5), 55.2
(
3
ꢂ
1
1
and 3-chloroiodobenzene (0.14 mL); m.p.: 93e95 C; H NMR
(400 MHz, DMSO‑d ): 2.54e2.56 (m, 1H), 2.94e3.01 (m, 1H),
3.13e3.18 (m, 1H), 3.41e3.53 (m, 2H), 7.07e7.13 (m, 3H), 7.24e7.29
m, 2H), 7.32e7.40 (m, 4H), 7.86 (AA'XX’, JAA’/XX’ ¼ 2.0 Hz,
AX ¼ 8.8 Hz, 2H), 7.93 (d, J ¼ 8.8 Hz, 2H), 8.17 (s, 1H), 10.70 (s, 1H).
(
(
C-7a), 138.6 (CeAr), 141.3 (C-3), 143.0 (CeAr), 149.9 (CeAr), 156.7
CeAr), 168.4 (CeAr), 191.5 (C]O).
6
d
(
2
1
.1.1.12. N-(4-Chlorophenyl)-4-(4-oxo-6-phenyl-4,5,6,7-tetrahydro-
H-indazol-1-yl)benzenesulfonamide (12). Compound 12 was ob-
J
1
3
6
C NMR (100 MHz, DMSO‑d ): d 31.0 (C-6), 41.6 (C-7), 44.9 (C-5),
tained as a solid (0.16 g, 35%) starting from compound 30b (0.34 g)
118.8 (CeAr), 119.8 (C-3a), 121.1 (2CeAr), 124.1 (2CeAr), 124.5
(CeAr), 127.4 (CeAr), 127.6 (2CeAr), 128.7 (CeAr), 129.0 (2CeAr),
131.5 (CeAr), 133.9 (CeAr), 138.5 (C-7a), 139.1 (CeAr), 139.5 (CeAr),
142.2 (C-3), 143.4 (CeAr), 150.4 (CeAr), 192.0 (C]O).
ꢂ
1
and 1-chloro-4-iodobenzene (0.27 g); m.p.: 100e102 C; H NMR
400 MHz, DMSO‑d ): 2.53e2.55 (m, 1H), 2.94e3.01 (m, 1H),
.13e3.16 (m, 1H), 3.41e3.53 (m, 2H), 7.11 (AA'XX’, JAA’/XX’ ¼ 2.0 Hz,
AX ¼ 8.8 Hz, 2H), 7.24e7.40 (m, 7H), 7.84e7.91 (m, 4H), 8.17 (s, 1H),
0.56 (s, 1H). 13C NMR (100 MHz, DMSO‑d
): 30.5 (C-6), 41.0 (C-7),
4.4 (C-5), 120.6 (C-3a), 121.8 (2CeAr), 123.4 (2CeAr), 126.8 (CeAr),
27.0 (2CeAr), 128.1 (2CeAr), 128.5 (2CeAr), 129.1 (2CeAr), 131.2
(
6
d
3
J
1
6
d
2.1.1.17. N-(3-Nitrophenyl)-4-(4-oxo-6-phenyl-4,5,6,7-tetrahydro-
1H-indazol-1-yl)benzenesulfonamide (17). Compound 17 was ob-
tained as a solid (0.28 g, 60%) starting from compound 30b (0.34 g)
4
1
ꢂ
1
(
(
CeAr), 134.4 (CeAr), 136.4 (CeAr), 138.6 (C-7a), 141.6 (C-3), 142.9
CeAr), 149.8 (CeAr), 191.4 (C]O).
and 1-iodo-3-nitrobenzene (0.28 g); m.p.: 105e107 C; H NMR
(400 MHz, DMSO‑d ): 2.54e2.55 (m, 1H), 2.93e3.01 (m, 1H),
6
d
4

S. Salerno, G. Amendola, A. Angeli et al.
European Journal of Medicinal Chemistry 220 (2021) 113490
ꢂ
1
3
.12e3.17 (m, 1H), 3.40e3.52 (m, 2H), 7.24e7.28 (m, 1H), 7.32e7.40
(0.30 g) and 1-iodo-4-nitrobenzene (0.28 g); m.p.: 222e224 C; H
NMR (400 MHz, DMSO‑d ): 1.02 (s, 6H), 2.38 (s, 2H), 3.00 (s, 2H),
(
m, 4H), 7.54e7.56 (m, 2H), 7.86e7.90 (m, 3H), 7.95e7.97 (m, 3H),
6
d
1
3
8
.17 (s,1H),11.05 (s,1H). C NMR (100 MHz, DMSO‑d
6
):
d
30.5 (C-6),
7.36 (AA'XX’, JAA’/XX’ ¼ 2.0 Hz, JAX ¼ 9.0 Hz, 2H), 7.89 (AA'XX’, JAA’/
4
(
1
1.0 (C-7), 44.4 (C-5), 113.6 (CeAr), 118.7 (CeAr), 120.6 (C-3a), 123.6
2CeAr), 125.5 (CeAr), 126.8 (CeAr), 127.0 (2CeAr), 128.3 (2CeAr),
28.5 (2CeAr), 130.9 (CeAr), 137.7 (CeAr), 138.7 (C-7a), 138.8
XX’ ¼ 2.0 Hz, JAX ¼ 8.8 Hz, 2H), 8.06 (AA'XX’, JAA’/XX’ ¼ 2.0 Hz,
J
J
AX ¼ 8.8 Hz, 2H), 8.13 (s, 1H), 8.17 (AA'XX’, JAA’/XX’ ¼ 2.0 Hz,
13
AX ¼ 9.2 Hz, 2H), 11.44 (s, 1H). C NMR (100 MHz, DMSO‑d
), 35.6 (C-6), 36.2 (C-7), 51.4 (C-5), 118.2 (2CeAr), 119.8 (C-3a),
123.9 (2CeAr),125.6 (2CeAr),128.4 (2CeAr),137.8 (CeAr),138.7 (C-
a), 142.0 (C-3), 142.8 (CeAr), 144.1 (CeAr), 149.5 (CeAr), 192.1 (C]
6
): d 27.7
(
(
CeAr), 141.8 (C-3), 142.9 (CeAr), 148.2 (CeAr), 149.9 (CeAr), 191.4
C]O).
(2CH
3
7
2
(
.1.1.18. 4-(4-Oxo-6-phenyl-4,5,6,7-tetrahydro-1H-indazol-1-yl)-N-
m-tolyl)benzenesulfonamide (18). Compound 18 was obtained as a
solid (0.11 g, 25%) starting from compound 30b (0.34 g) and 3-
O).
2.1.1.23. 4-(6,6-Dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-
yl)-N-(4-tolyl)benzenesulfonamide (23). Compound 23 was ob-
tained as a solid (0.12 g, 30%) starting from compound 30c (0.30 g)
ꢂ
1
iodotoluene (0.14 mL); m.p.: 87e89 C; H NMR (400 MHz,
DMSO‑d ): 2.17 (s, 3H), 2.54e2.55 (m, 1H), 2.94e3.01 (m, 1H),
.11e3.15 (m, 1H), 3.40e3.53 (m, 2H), 6.83e6.85 (m, 1H), 7.08e7.12
6
d
ꢂ
1
3
and 1-iodo-4-methylbenzene (0.25 g); m.p.: 205e207 C; H NMR
(400 MHz, DMSO‑d ): 1.02 (s, 6H), 2.19 (s, 3H), 2.37 (s, 2H), 2.97 (s,
2H), 7.01 (d, J ¼ 8.8 Hz, 2H), 7.06 (d, J ¼ 8.8 Hz, 2H), 7.81 (d,
(
m, 1H), 7.25e7.27 (m, 1H), 7.32e7.40 (m, 4H), 7.83 (d, J ¼ 8.8 Hz,
6
d
13
2
H), 7.90 (d, J ¼ 8.8 Hz, 2H), 8.17 (s, 1H), 10.34 (s, 1H). C NMR
): 21.1 (CH ), 30.6 (C-6), 41.1 (C-7), 44.5 (C-5),
17.4 (CeAr), 120.6 (CeAr), 120.8 (C-3a), 123.6 (2CeAr), 125.1
CeAr), 127.0 (CeAr), 127.2 (2CeAr), 128.3 (2CeAr), 128.6 (2CeAr),
29.1 (CeAr), 137.4 (CeAr), 138.6 (C-7a), 138.6 (CeAr), 138.7 (CeAr),
13
(
100 MHz, DMSO‑d
6
d
3
J ¼ 8.8 Hz, 2H), 7.90 (d, J ¼ 8.8 Hz, 2H), 8.11 (s, 1H), 10.26 (s, 1H).
C
1
(
1
NMR (100 MHz, DMSO‑d ): 20.5 (CH ), 27.8 (2CH ), 35.6 (C-6),
6
d
3
3
36.1 (C-7), 51.4 (C-5), 119.8 (C-3a), 121.0 (2CeAr), 123.7 (2CeAr),
128.4 (2CeAr), 129.9 (2CeAr), 133.9 (CeAr), 134.9 (CeAr), 138.6 (C-
7a), 138.7 (CeAr), 141.5 (C-3), 149.5 (CeAr), 192.3 (C]O).
1
41.5 (C-3), 143.0 (CeAr), 149.9 (CeAr), 191.6 (C]O).
2
.1.1.19. 4-(6,6-Dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)-
2.1.1.24. 4-(6,6-Dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-
yl)-N-(3-methoxyphenyl)benzenesulfonamide (24). Compound 24
was obtained as a solid (0.10 g, 25%) starting from compound 30c
N-phenylbenzenesulfonamide (19). Compound 19 was obtained as a
solid (93 mg, 25%) starting from compound 30c (0.30 g) and
ꢂ
1
ꢂ
1
iodobenzene (0.13 mL); m.p.: 212e214 C; H NMR (400 MHz,
DMSO‑d ):
1.02 (s, 6H), 2.36 (s, 2H), 2.98 (s, 2H), 7.05 (t, J ¼ 7.4 Hz),
.13e7.15 (m, 2H), 7.26 (m, 2H), 7.83 (AA'XX’, JAA’/XX’ ¼ 2.0 Hz,
AX ¼ 8.8 Hz, 2H), 7.93 (AA'XX’, JAA’/XX’ ¼ 2.0 Hz, JAX ¼ 8.8 Hz), 8.11 (s,
H), 10.44 (s, 1H). 13C NMR (100 MHz, DMSO‑d
): 27.7 (2CH ), 35.5
(0.30 g) and 3-iodoanisole (0.13 mL); m.p.: 204e206 C; H NMR
(400 MHz, DMSO‑d ): 1.02 (s, 6H), 2.37 (s, 2H), 2.98 (s, 2H), 3.67 (s,
6
d
6
d
7
J
1
3H), 6.61e6.64 (m, 1H), 6.71e6.73 (m, 2H), 7.16 (t, J ¼ 8.2 Hz, 1H),
7.84 (d, J ¼ 8.8 Hz, 2H), 7.95 (d, J ¼ 8.8 Hz, 2H), 8.11 (s, 1H), 10.47 (s,
13
6
d
3
1H). C NMR (100 MHz, DMSO‑d
6
):
d
27.7 (2CH
3
), 35.5 (C-6), 36.0
(
(
1
C-6), 36.0 (C-7), 51.4 (C-5), 119.7 (C-3a), 120.2 (2CeAr), 123.6
2CeAr), 124.4 (CeAr), 128.3 (2CeAr), 129.3 (2CeAr), 137.5 (CeAr),
38.5 (C-7a), 138.6 (CeAr), 141.5 (C-3), 149.4 (CeAr), 192.1 (C]O).
3
(C-7), 51.4 (C-5), 55.1 (OCH ), 105.9 (CeAr), 109.4 (CeAr), 112.1
(CeAr), 119.7 (C-3a), 123.7 (2CeAr), 128.3 (2CeAr), 130.2 (CeAr),
138.5 (CeAr), 138.6 (C-7a), 138.7 (CeAr), 141.6 (C-3), 149.4 (CeAr),
159.8 (CeAr), 192.1 (C]O).
2
.1.1.20. 4-(6,6-Dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-
yl)-N-(4-methoxyphenyl)benzenesulfonamide (20). Compound 20
was obtained as a solid (0.10 g, 25%) starting from compound 30c
2.1.1.25. N-(3-Chlorophenyl)-4-(6,6-dimethyl-4-oxo-4,5,6,7-
tetrahydro-1H-indazol-1-yl)benzenesulfonamide (25).
ꢂ
1
(
(
3
0.30 g) and 4-iodoanisole (0.26 g); m.p.: 98e100 C; H NMR
400 MHz, DMSO‑d ): 1.02 (s, 6H), 2.37 (s, 2H), 2.98 (s, 2H), 3.67 (s,
H), 6.82 (AA'XX’, JAA’/XX’ ¼ 2.2 Hz, JA ¼ 8.8 Hz, 2H), 7.02 (AA'XX’,
Compound 25 was obtained as a solid (0.16 g, 40%) starting from
compound 30c (0.30 g) and 3-chloroiodobenzene (0.14 mL); m.p.:
6
d
ꢂ
1
X
200e202 C; H NMR (400 MHz, DMSO‑d
6
): d 1.02 (s, 6H), 2.37 (s,
J
8
AA’/XX’ ¼ 2.4 Hz, JAX ¼ 9.0 Hz, 2H), 7.83 (dd, J ¼ 8.8 Hz, J ¼ 9.2 Hz, 4H),
2H), 2.99 (s, 2H), 7.11e7.16 (m, 3H), 7.28e7.32 (m, 1H), 7.86 (d,
1
3
13
.11 (s, 1H), 10.08 (s, 1H). C NMR (100 MHz, DMSO‑d
), 35.6 (C-6), 36.1 (C-7), 51.5 (C-5), 55.3 (OCH ), 114.5 (2CeAr),
19.7 (C-3a), 123.6 (2CeAr), 123.7 (2CeAr), 128.3 (2CeAr), 129.9
CeAr), 138.5 (C-7a), 138.6 (CeAr), 141.4 (C-3), 149.5 (CeAr), 156.8
CeAr), 192.2 (C]O).
6
):
d
27.8
J ¼ 8.8 Hz, 2H), 7.96 (d, J ¼ 8.8 Hz, 2H), 8.12 (s, 1H), 10.74 (s, 1H).
C
(
2CH
3
3
NMR (100 MHz, DMSO‑d ): 27.7 (2CH ), 35.5 (C-6), 36.1 (C-7), 51.4
6
d
3
1
(
(
(C-5), 118.3 (CeAr), 119.3 (CeAr), 119.8 (C-3a), 123.7 (2CeAr), 124.1
(CeAr), 128.3 (2CeAr), 131.1 (CeAr), 133.5 (CeAr), 138.2 (CeAr),
138.5 (C-7a), 139.1 (CeAr), 141.7 (C-3), 149.4 (CeAr), 192.1 (C]O).
2
.1.1.21. N-(4-Chlorophenyl)-4-(6,6-dimethyl-4-oxo-4,5,6,7-
2.1.1.26. 4-(6,6-Dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-
yl)-N-(3-nitrophenyl)benzenesulfonamide (26). Compound 26 was
obtained as a solid (0.12 g, 30%) starting from compound 30c
tetrahydro-1H-indazol-1-yl)benzenesulfonamide (21).
Compound 21 was obtained as a solid (0.20 g, 50%) starting from
compound 30c (0.30 g) and 1-chloro-4-iodobenzene (0.27 g); m.p.:
ꢂ
1
(0.30 g) and 1-iodo-3-nitrobenzene (0.28 g); m.p.: 210e212 C; H
NMR (400 MHz, DMSO‑d ): 1.01 (s, 6H), 2.36 (s, 2H), 2.97 (s, 2H),
7.58e7.59 (m, 2H), 7.86 (d, 2H, J ¼ 8.8 Hz), 7.90e7.93 (m, 1H),
ꢂ
1
1
23e125 C; H NMR (400 MHz, DMSO‑d
H), 2.98 (s, 2H), 7.15 (AA'XX’, JAA’/XX’ ¼ 2.0 Hz, JAX ¼ 8.8 Hz, 2H), 7.33
AA'XX’, JAA’/XX’ ¼ 2.0 Hz, JAX ¼ 8.8 Hz, 2H), 7.84 (AA'XX’, JAA’/
XX’ ¼ 2.0 Hz, JAX ¼ 8.8 Hz, 2H), 7.93 (AA'XX’, JAA’/XX’ ¼ 2.4 Hz,
6
):
d
1.02 (s, 6H), 2.37 (s,
6
d
2
(
13
7.97e8.00 (m, 3H), 8.11 (s, 1H), 11.07 (s, 1H). C NMR (100 MHz,
DMSO‑d ): 27.8 (2CH ), 35.6 (C-6), 36.1 (C-7), 51.4 (C-5), 113.8
6
d
3
13
J
AX ¼ 9.0 Hz, 2H), 8.11 (s, 1H), 10.59 (s, 1H). C NMR (100 MHz,
DMSO‑d ): 27.8 (2CH ), 35.6 (C-6), 36.1 (C-7), 51.5 (C-5), 119.8 (C-
a), 121.9 (2CeAr), 122.7 (2CeAr), 128.3 (2CeAr), 128.6 (CeAr),
29.4 (2CeAr), 136.5 (CeAr), 138.3 (C-7a), 138.6 (CeAr), 141.7 (C-3),
49.5 (CeAr), 192.2 (C]O).
(CeAr), 118.8 (CeAr), 119.8 (C-3a), 123.8 (2CeAr), 125.8 (CeAr),
128.4 (2CeAr), 131.1 (CeAr), 137.9 (CeAr), 138.6 (C-7a), 139.0
(CeAr), 141.9 (C-3), 148.3 (CeAr), 149.5 (CeAr), 192.1 (C]O).
6
d
3
3
1
1
2.1.1.27. 4-(6,6-Dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-
yl)-N-(3-tolyl)benzenesulfonamide (27). Compound 27 was ob-
tained as a solid (0.13 g, 35%) starting from compound 30c (0.30 g)
2
.1.1.22. 4-(6,6-Dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-
ꢂ
1
yl)-N-(4-nitrophenyl)benzenesulfonamide (22). Compound 22 was
obtained as a solid (0.10 g, 25%) starting from compound 30c
and 3-iodotoluene (0.14 mL); m.p.: 216e218 C; H NMR (400 MHz,
DMSO‑d ): 1.02 (s, 6H), 2.21 (s, 3H), 2.37 (s, 2H), 2.98 (s, 2H),
6
d
5

S. Salerno, G. Amendola, A. Angeli et al.
European Journal of Medicinal Chemistry 220 (2021) 113490
6
.86e6.87 (m, 1H), 6.93e6.95 (m, 2H), 7.13 (t, 1H, J ¼ 8.0), 7.82 (d,
The zinc-specific non-bonded pairwise potentials were included in
the creation of the grid parameter file. A set of grids of
13
J ¼ 8.8 Hz, 2H), 7.93 (d, J ¼ 8.8 Hz, 2H), 8.11 (s, 1H), 10.37 (s, 1H).
C
NMR (100 MHz, DMSO‑d
6
):
d
21.1 (CH
3
), 27.8 (2CH
3
), 35.6 (C-6), 36.1
50 Å ꢁ 50 Å ꢁ 50 Å with 0.375 Å spacing was calculated considering
the docking area for all the ligands atom types employing Auto-
Grid4. For every possible compound 11 conformation/configuration
state, 100 independent docking simulations were achieved. Each
docking calculation comprised 20 million energy evaluations
employing the Lamarckian genetic algorithm local search (GALS)
method. This latter assesses a population of viable docking solu-
tions and propagates the best individuals from each generation into
the following generation of feasible solutions. A low-frequency
local search according to the method of Solis and Wets was
applied to every docking attempt to guarantee that the final solu-
tion represented a local minimum. All dockings were performed
with a population size of 250, and 300 iterations of Solis and Wets
local search were applied with a probability of 0.06. A rate of mu-
tation of 0.02 and a crossover rate of 0.8 were used to produce new
docking attempts for following generations, and the best individual
from each generation was propagated over the following genera-
tion. The docking results from every (100) independent docking
calculation were clustered based on the root-mean-square devia-
tion (rmsd) (solutions differing by less than 2.0 Å) between the
Cartesian coordinates of the atoms and were ranked based on the
(
(
1
1
C-7), 51.4 (C-5), 117.3 (CeAr), 119.7 (C-3a), 120.8 (CeAr), 123.7
2CeAr), 125.1 (CeAr), 128.3 (2CeAr), 129.2 (CeAr), 137.5 (CeAr),
38.5 (CeAr), 138.7 (C-7a), 138.7 (CeAr), 141.5 (C-3), 149.5 (CeAr),
92.2 (C]O).
2
.2. CA inhibition assays
An Applied Photophysics stopped-flow instrument was used for
2
assaying the CA catalyzed CO hydration activity [18]. Phenol red (at
a concentration of 0.2 mM) was used as an indicator, working at the
absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) as a
buffer, and 20 mM Na
strength), following the initial rates of the CA-catalyzed CO
dration reaction for a period of 10e100 s. The CO concentrations
2
SO
4
(for maintaining constant ionic
2
hy-
2
ranged from 1.7 to 17 mM for the determination of the kinetic
parameters and inhibition constants [6]. For each inhibitor, at least
six traces of the initial 5e10% of the reaction were used for deter-
mining the initial velocity. The uncatalyzed rates were determined
in the same manner and subtracted from the total observed rates.
Stock solutions of the inhibitor (0.1 mM) were prepared in
distillededeionized water and dilutions up to 0.01 nM were done
thereafter with the assay buffer. Inhibitor and enzyme solutions
were preincubated together for 15 min at room temperature before
the assay, to allow for the formation of the EeI complex. The in-
hibition constants were obtained by non-linear least-squares
methods using PRISM 3 and the Cheng-Prusoff equation as re-
ported earlier and represent the mean from at least three different
determinations. All CA isoforms were recombinant proteins ob-
tained in-house, as reported earlier [19e21]. Enzymes concentra-
tions in the assay system ranged between 5 and 12 nM.
free energy of binding (
selected among the four conformation/configuration states docking
results factoring in the AD4, docking cluster population size, and
DGAD4). Compound 11 binding pose was
DG
the existing structure-activity relationship.
2.3.2. QM calculations
Compounds 12 and 13 were virtually prepared according to the
2D Sketcher/LigPrep protocol detailed above for compound 11. The
electrostatic potentials of compounds 11, 12, 13 were calculated
using the Single Point Energy tool using default settings within the
Jaguar software [29] and mapped onto their electron density. To
display the electron density surface the isovalue of 0.001 electron/
Bhor3 was chosen, using the calculated electrostatic potential as a
rainbow color scheme, with a range of values from ꢀ0.15 kcal/mol
2
2
.3. Molecular modeling studies
.3.1. Molecular docking
The latest version of the AD4 docking software (version 4.2) [22]
a
(red) to 0.01 kcal/mol (purple). The pK of the benzenesulfonamide
nitrogen of compounds 11, 12, 13 was also calculated through the
together with its GUI AutoDockTools (ADT) and the AutoDock4(Zn)
force field [23] were employed. The hCA II X-ray structure used for
the experiment had the PDB code 3K34 [24]. The protein structure
was prepared for the docking using the Protein Preparation Wizard
of the Maestro suite [25] that adds bond orders and hydrogen
atoms, deletes water molecules, and produces the appropriate
protonation states. The co-crystal ligand of 3K34 was separated
from the cognate protein. The 2D Sketcher tool of Maestro was used
to build compound 11. The ligand was considered in all its possible
configurations (R, S) and conformations (two tetrahydro-
indazolone scaffold possible flips): four in total. For each of them
the appropriate protonation and tautomeric metal-binding state, as
well as their optimized geometry, was obtained through LigPrep,
part of the same suite. Through Maestro, the X-ray structures of
hCA I (PDB 6EVR) [26], hCA IV (PDB 5JN9) [27], and hCA IX (PDB
Jaguar software using standard settings.
3. Results and discussion
3.1. Synthetic approach
The synthesis of compounds 1e27 is reported in Scheme 1. The
key intermediates were the bicyclic tetrahydroindazoles 30a-c,
which were prepared as previously reported by us [16]. Briefly, the
treatment of the commercially available 5-substituted-1,3-
cyclohexandiones 28a-c with an excess of N,N-dimethylforma-
ꢂ
mide-dimethylacetale (DMF-DMA) at 100 C for 1 h furnished the
intermediates 29a¡c (Scheme 1) in quantitative yield.
Then, compounds 29a¡c were reacted with a slight excess of 4-
ꢂ
5
FL6) [28], were downloaded and superimposed on the structure of
hCA II. The ligands were translated in the AD4 specific file format
PDBQT) using the python scripts prepare_ligand4.py and prepar-
hydrazinobenzenesulfonamide hydrochloride in ethanol at 80
C
for 5e20 h (TLC analysis), to obtain derivatives 30a¡c. Finally, the
(
new indazolyl-N-arylbenzenesulfonamides 1e27 were obtained by
e_receptor4.py, part of ADT, applying the standard settings.
Following the AutoDock4(Zn) force field protocol, to add the
tetrahedral zinc pseudo atoms to the receptor PDBQT the script
zinc_pseudo.py, part of the material provided with the force field,
was employed. The docking area was centered on the active site.
reacting the bicyclic tetrahydroindazoles 30a-c with the proper
0
phenyliodide in acetonitrile (MeCN) in the presence of K
2
CO
3
, N,N -
dimethylethylendiamine (DMEDA) and a catalytic amount of CuI.
ꢂ
The reaction mixture was heated at 100 C and stirred for 8e24 h
(TLC analysis); after cooling, the suspension obtained was diluted
6

S. Salerno, G. Amendola, A. Angeli et al.
European Journal of Medicinal Chemistry 220 (2021) 113490
with water, acidified with 2.0 N HCl to pH ¼ 4, and extracted with
EtOAc. The organic phase dried, filtered, and evaporated under
reduced pressure furnished the crude material which was purified
by flash chromatography, yielding the pure target derivatives 1e27.
biological activity of the indazolyl-N-arylbenzenesulfonamides
1e27 appears strictly dependent on the substitution pattern at 6-
and R
and R
3
-position, suggesting an interdependent effect of the R
groups on the inhibitory profile of this series of congeneric
1 2
, R ,
3
derivatives. Specifically, the unsubstituted compounds (1e9) show
good selectivity towards the hCA I with respect to the other iso-
3
.2. In vitro CA inhibition assay and structure-activity relationships
forms tested, and good potency, with K
to 6.3 M. This trend appears to be independent from the decora-
tion of the pendant phenyl ring (R ), with the sole exception of
compound 6 (R ), which results scarcely active against
¼ m-OCH
all the tested hCAs.
The presence of a 6-phenyl (compounds 10e18) determined a
decrease in hCA I inhibition potency (K values ranging from 8.6
to 69.1 M) and a gain in activity toward the hCA II isoform (K
values between 0.41 M and 23.6 M), with respect to their 6-
unsubstituted analogs 1e9. The presence of R , Cl (com-
¼ OCH
i
values ranging from 1.4 mM
(
SARs)
m
3
All the newly synthesized compounds 1e27 were investigated
3
3
for their enzyme inhibitory ability against four physiologically
relevant CA isoforms, the human hCA I, II, IV, and IX (Table 1), by a
stopped-flow CO
2
hydrase assay. Acetazolamide (AAZ, 5-
mM
i
acetamido-1,3,4-thiadiazole-2-sulfonamide) was used as the stan-
dard drug in the assay [18]. Data referring to the primary sulfon-
amide parent compounds V a-c [16] were also reported in Table 1
for comparison purposes.
Results listed in Table 1 evidence that, in general, the insertion of
a secondary sulfonamide function on the tetrahydroindazole scaf-
fold produced inhibitors (1e27) with a moderate micromolar po-
tency, lower with respect to their primary sulfonamide parent
compounds (V a-c). Fig.1 schematizes the main SARs data achieved.
In particular, all the compounds 1e27 resulted in weak inhibi-
tion of the hCA IV and hCA IX isoforms, with the sole exception of
m
i
m
m
3
3
pounds 11, 12) at para-position of the pendant phenyl ring seemed
to produce a positive effect on hCA II inhibitory potency. The best
performing compound of this subseries is indeed represented by p-
methoxyphenyl-substituted derivative 11, that is a potent (K
.41
M) and selective (CAI/CAII ¼ 21, CAIV/CAII ¼ 153, CAIX/
i
0
m
CAII ¼ 97.6) hCA II inhibitor.
Mixed results towards hCA II are associated with the presence of
the two methyl groups at the 6-position of the heterocyclic scaffold
13 that shows submicromolar potency towards hCA IV, moderate
(compounds 19e27), which should be correlated with the nature
selectivity vs hCA II and hCA IX, and high selectivity vs hCA I. For
what concerns the hCA I and hCA II enzymes, interestingly, the
and position of the group on the pendant phenyl ring (R ). A pair-
3
Scheme 1. Synthesis of compounds 1-27.
7

S. Salerno, G. Amendola, A. Angeli et al.
European Journal of Medicinal Chemistry 220 (2021) 113490
Fig. 1. Schematic representation of the main SARs data achieved in the new series of indazolyl-N-arylbenzenesulfonamides.
wise comparison of the R
,R -dimethyl substituted one (19e27), outlined that similar or
lower
derivatives 19, 22, 25, 26, 27 (R
values of 26.5 M, 34.8
1 2
,R -unsubstituted subclass (1e9) with the
R
1
2
hCAII
inhibitory
potency
is
, m-Cl, m-NO
M, >100
values of 31.0
M, respectively). Conversely,
¼p-OCH , p-Cl, p-CH , m-OCH
respectively) show a moderately higher hCAII inhibitory activity
values of 2.6 M, 3.5 M, 4.0 M, 4.1 M, respectively) with
respect to their 6-unsubstituted counterparts 2, 3, 5, and 6 (K
values of 5.9 M, 27.0 M, 8.4 M, >100 M, respectively), and low
potency towards the other isoforms tested (with the only exception
observed
, m-CH
M,
for
3
,
m
3
¼ H, p-NO
2
2
K
i
m
m
M, >100
m
M, 57.9
m
respectively) with respect to the analogs 1, 4, 7, 8, 9 (K
M, 50.3 M, 21.2 M, >100 M, 35.1
compounds 20, 21, 23, and 24 (R
i
m
m
m
m
m
3
3
3
3
,
(
K
i
m
m
m
m
i
m
m
m
m
i
of 21, hCAI K 9.1mM), thus resulting in good CAII-selective
inhibitors.
3.3. Molecular modelling studies
To elucidate the reasons behind the SARs data achieved for the
newly designed compounds, a series of in silico studies were
accomplished on compound 11 which demonstrated to be
comparatively the most potent and hCA II selective inhibitor of the
whole set. The AutoDock Zinc [24] docking protocol, specific for
zinc-chelating ligands, was employed as implemented in our pre-
vious studies on hCAs inhibitors [16,30]. In this inspection the
structure of the hCA II having PDB code 3K34 was used. Together
with 11, the co-crystal ligand of the aforementioned structure was
also considered for re-docking experiments that were attained to
probe the predictive potential of the employed protocol. Indeed,
AD4 was able to predict a binding pose of the complexed ligand
within 1.02 Å RMSD from the experimental one with a predicted
Fig. 2. Compound 11 docked pose within the hCA II binding site (PDB 3K34). The
protein is depicted in salmon ribbons and sticks. Important residues are labeled. The
ligand is depicted in cyan sticks. The catalytic zinc ion is represented in violet. The
metal chelation bonds are represented as purple dashed lines.
p-p interactions are
shown as green dashed lines. The H-bond is represented as a red dashed line. The
picture was rendered using the UCSF Chimera software [31].
chain. Indeed, the loss of said interaction might account for the
lower hCA II inhibitory power displayed by the compounds devoid
of an H-bond acceptor group at the p-position (10, 14, 15e18).
SARs data indicate that, while still able to engage in favorable
binding free energy (
DGAD4) of ꢀ6.72 kcal/mol. Encouraged by
these results we analyzed the docking solution achieved for com-
2 3
ligand-enzyme contacts, the p-Cl and p-NO substitutions in R
pound 11 R-enantiomer in the hCA II binding site obtaining a pre-
have a detrimental effect on the inhibition potency against hCA II
(compounds 12 and 13, respectively). To better profile the influence
of the benzenesulfonamide substitution pattern on the sulfon-
amide nitrogen acidity, single-point energy (SPE) and pKa quantum
mechanics (QM) calculations were performed on compounds 11,12,
and 13. At first, through the Jaguar QM software [29], part of the
dicted
D
G
AD4 of ꢀ7.27 kcal/mol for the most populated solution
(
Fig. 2). Firstly, the secondary sulfonamide is predicted to chelate
the catalytic zinc ion through its negatively charged nitrogen in a
geometry that is consistent with other CAIs. Moreover, one of the
sulfonamide oxygen atoms accepts an H-bond from the T200
backbone NH.
Schr
o€ dinger molecular modeling suite, the electrostatic potential
The phenyl ring attached to the sulfonamide nitrogen is lodged
in a cleft lined by the polar side chains of the residues N62, N67,
Q92, H94, and T200. Here, the ligand aromatic moiety engages in a
pattern of charge-transfer interactions with the side chains amide
mapped on the electron density surface was calculated for each of
the ligands (Fig. 3). The SPE QM analysis reveals that 11, bearing an
electron-donating p-methoxy group, shows a more substantial
negative partial charge on the sulfonamide nitrogen than both
compounds 12 and 13, which feature at the same position electron-
withdrawing moieties (chlorine and nitro group, respectively).
p
-faces of N62, N67, Q92, and with the H94 imidazole ring. Of note,
the phenyl ring p-methoxy substitution should be in a favorable
position to accept an additional H-bond from the N62 amide side
2
Indeed, compound 12 p-NO strong electron-withdrawing effect
8

S. Salerno, G. Amendola, A. Angeli et al.
European Journal of Medicinal Chemistry 220 (2021) 113490
Fig. 3. QM-derived electrostatic potential mapped on the electron density surface for compounds 11, 12, and 13. To display the electron density surface the isovalue of 0.001
3
electron/Bhor was chosen, using the calculated electrostatic potential as a rainbow color scheme, with a range of values from ꢀ0.15 kcal/mol (red) to 0.01 kcal/mol (purple).
resulted in the weakest negative electrostatic potential on the
sulfonamide nitrogen among the three inhibitors considered. A
similar trend could also be observed with the results of the pKa QM
calculations always carried out with the Jaguar software. In this in
silico test, 11 reported a pKa value for the benzenesulfonamide
nitrogen of 8.3, 12 reported a value of 7.1, while 13 yielded a 4.7
value. In conclusion, the QM calculations confirm that the benze-
nesulfonamide substitution pattern influences the nitrogen acidity,
which would in turn affect the zinc chelation strength, with the p-
methoxy group being the most favorable substitution.
As depicted in Fig. 1, the phenyl ring linking the benzenesulfo-
namide and the tetrahydro-indazolone scaffold would form van der
Waals's contacts with the nearby sidechains of L198, T200, and
P202. As for the tetrahydro-indazolone ring and its 6-phenyl sub-
stituent, they should fill the partially hydrophilic cleft comprising
the residues N67, E69, I91, Q92, and F131. In particular, the
Fig. 4. 11 hCA II docked binding pose within the hCA I (PDB 6EVR) (A and D), hCA IV (PDB 5JN9) (B and E), and hCA IX (PDB 5FL6) (C and F) structure. The ligand is represented as
cyan stcks and transparent surface. The CAs structure is depicted as green, yellow, and purple sticks and surfaces for hCA I, hCA I, and hCA IX, respectively. Steric clashes are
explicitly reported in panels DeF as red lines. The pictures were rendered using the UCSF ChimeraX (AeC) [34] and Chimera (DeF) software [31].
9

S. Salerno, G. Amendola, A. Angeli et al.
European Journal of Medicinal Chemistry 220 (2021) 113490
tetrahydro-indazolone would form an edge-to-face aryl-aryl
interaction with the F131 sidechain. While the 6-phenyl ring should
engage in van der Waals contacts with the rest of the residues in the
all these data could prospectively guide the future design of more
active and isoform-selective CAIs.
cleft, it would also benefit from the interactions with the amide
p
-
Declaration of competing interest
faces of the nearby N67 and Q92. It could then be inferred that
compounds lacking the phenyl ring at the 6-position (1e9, 19e27)
should lose the aforementioned favorable interactions with the
receptor.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Besides, the cavity filled by the tetrahydro-indazolone scaffold
and the 6-phenyl ring is adjacent and partially overlapping with the
so-called "selectivity hotspot" of the hCA II binding site. Said hot-
spot, comprising the residues 125e137, was described as a highly
variable sequence among all the CAs catalytic sites, thus playing a
significant role in determining ligand isoform selectivity [32].
Furthermore, Pinard et al. also reported in their work on CAs that
residues N67 and I91 are also scarcely conserved among the
different isoforms [33]. As a result, bulky substitutions at the 6-
position, such as a phenyl ring, might directly influence the iso-
form selectivity. To acquire further insights on the reasons for the
selectivity displayed by the novel inhibitors, the binding pose ob-
tained for compound 11 on hCA II was rigidly translated in the
binding sites of hCA I, hCA IV, and hCA IX (Fig. 4). In this orientation,
Acknowledgments
This research was funded by the University of Campania Luigi
Vanvitelli (VALERE: Vanvitelli per la Ricerca, ANIMA and VALER-
EPlus projects) and Campania and Regional Government Technol-
ogy Platform Lotta alle Patologie Oncologiche: iCURE to S.C, by the
University of Pisa (Code PRA_2020_58) to A.M.M. and by the PRIN
2017 project (Code 2017XYBP2R) of the Italian MIUR to A.M.M.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113490.
11 would give rise to multiple steric clashes in each of the isoforms.
In particular, by adopting the binding orientation responsible for
References
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prevent the correct orientation of 11 is M67 which corresponds to
N67 of hCA II. Lastly, in hCA IX the steric clash with Q71, the loss of a
charge transfer interaction (F131 of hCA II mutated with V130 of
hCA IX), and a general relocation of the other residues lining the
enzyme binding site should be the cause for the lower inhibition of
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