Synthesis and cytotoxic activity of chiral sulfonamides based on the 2-azabicycloalkane skeleton

Article abstract of DOI:10.3390/molecules25102355

A series of chiral sulfonamides containing the 2-azabicycloalkane scaffold were prepared from aza-Diels–Alder cycloadducts through their conversion to amines based on 2-azanorbornane or the bridged azepane skeleton, followed by the reaction with sulfonyl chlorides. The cytotoxic activity of the obtained bicyclic derivatives was evaluated using human hepatocellular carcinoma (HCC), medulloblastoma (MB), and glioblastoma (GBM) cell lines. Chosen compounds were shown to notably reduce cell viability as compared to nonmalignant cells.

Full text of DOI:10.3390/molecules25102355

molecules
Article
Synthesis and Cytotoxic Activity of Chiral Sulfonamides
Based on the 2-Azabicycloalkane Skeleton
Mahzeiar Samadaei 1 , Matthias Pinter 1 , Daniel Senfter 1 , Sibylle Madlener 1
,
Nataliya Rohr-Udilova 1,
*
, Dominika Iwan 2 , Karolina Kamin´ska 2
,
Elz˙bieta Wojaczyn´ska 2,
*
, Jacek Wojaczyn´ski 3 and Andrzej Kochel 3
1
Department of Internal Medicine III, Medical University of Vienna, AKH Vienna Währinger Gürtel 18-20,
1090 Vienna, Austria; mahzeiar.samadaei@meduniwien.ac.at (M.S.); matthias.pinter@meduniwien.ac.at (M.P.);
daniel.senfter@meduniwien.ac.at (D.S.); sibylle.madlener@meduniwien.ac.at (S.M.)
Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzez˙e Wyspian´skiego 27,
50-370 Wrocław, Poland; dominika.iwan@pwr.edu.pl (D.I.); karolina.kaminska@pwr.edu.pl (K.K.)
Faculty of Chemistry, University of Wrocław, F. Joliot-Curie St. 14, 50-383 Wrocław, Poland;
jacek.wojaczynski@chem.uni.wroc.pl (J.W.); andrzej.kochel@chem.uni.wroc.pl (A.K.)
Correspondence: nataliya.rohr-udilova@meduniwien.ac.at (N.R.-U.);
2
3
*
elzbieta.wojaczynska@pwr.edu.pl (E.W.); Tel.: +43-14040047230 (N.R.-U.); +48-713202410 (E.W.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 25 March 2020; Accepted: 11 May 2020; Published: 18 May 2020
Abstract: A series of chiral sulfonamides containing the 2-azabicycloalkane scaffold were prepared
from aza-Diels–Alder cycloadducts through their conversion to amines based on 2-azanorbornane
or the bridged azepane skeleton, followed by the reaction with sulfonyl chlorides. The cytotoxic
activity of the obtained bicyclic derivatives was evaluated using human hepatocellular carcinoma
(HCC), medulloblastoma (MB), and glioblastoma (GBM) cell lines. Chosen compounds were shown
to notably reduce cell viability as compared to nonmalignant cells.
Keywords: 2-azabicycloalkane; chiral sulfonamide; cytotoxic activity
1. Introduction
A continuous demand for novel, potent, and selective anticancer drugs has led to the development
of various classes of therapeutics. Among them, sulfonamides have attracted considerable attention
due to their high and multidirectional activity, and are used or tested as pharmaceutical agents,
possessing antibacterial, antiviral, anticancer, antimalarial, diuretic, hypoglycemic, anticonvulsant,
antirheumatic, and antithyroid properties [
structural motifs are also associated with impressive biological activity. For example, the biphenyl
moiety is present in numerous derivatives that have been tested in antiproliferative studies [ ].
The trifluoromethyl group is also regarded as a valuable pharmacophore [ ], which is found in marketed
drugs (e.g., nonsteroidal antiandrogen flutamide) and newly developed derivatives [10]. Additionally,
2-azanorbornane (2-azabicyclo[2.2.1]heptane) (Figure 1), an intrinsically chiral compound with
1–5]. In addition to functional groups, certain characteristic
6–8
9
1
a stable bicyclic skeleton, has been recognized as a versatile scaffold for asymmetric synthesis
and a precursor or a component of molecules exhibiting biological activity [11]. This system can
be prepared via the stereoselective aza-Diels–Alder reaction of chiral imines and cyclopentadiene
in both enantiomeric forms, even in large quantities (grams or kilograms) [12
of 2-azanorbornane have already been applied in various enantioselective transformations [11
–
17]. Derivatives
18].
,
An expanded bicyclic system, namely 2-azabicyclo[3.2.1]octane, can be prepared in a stereoselective
manner from 2-aznanorbornylmethanols under nucleophilic substitution reaction conditions [19].
Using this approach, isomeric amines based on the bridged piperidine (compound 2, Figure 1) or
Molecules 2020, 25, 2355; doi:10.3390/molecules25102355
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2355
2 of 10
azepane (isomer
3
, Figure 1) were prepared [20
,21]. We used these amines and their epimers for the
construction of bifunctional catalysts [22]. Monomeric and dimeric derivatives modified with amino
functions were also prepared and were found to inhibit the growth of selected tumor cell lines [7].
Figure 1. The 2-azanorbornane (1) and isomeric amines containing bicyclic skeletons (2,3).
The promising results of our study on the biological activity of amines bearing chiral azabicyclic
skeletons prompted us to prepare a series of corresponding sulfonamides. Based on literature reports,
we were convinced that introduction of the sulfonamide function, also with biphenyl substituents,
should result in the enhancement of cytotoxicity and bioavailability. For example, combinations of
sulfonamide and thiourea derivatives produced promising anticancer agents in the study by Ghorab et
al. [
2], while the methanesulfonamide analogue of cryptopleurine exhibited improved bioavailability
and antitumor activity, as reported by Kwon et al. [4]. In this article, we present the synthesis of
new bicyclic derivatives and the study of their interaction with selected human tumor cell lines and
nonmalignant primary cells. We believed that molecules that contain both sulfonamide and biphenyl
or trifluoromethyl moieties could lead to new hybrid architectures with improved biological profiles.
2. Materials and Methods
2.1. Instrumentation
Melting points were determined on the Schmelzpunkt Bestimmer Apotec melting point apparatus
(WEPA Apothekenbedarf GmbH & Co. KG., Hillscheid, Germany) using the standard open capillary.
1H and 13C NMR spectra were collected on Jeol 400yh (Joel Ltd., Tokyo, Japan), Bruker Avance III
500, and Bruker Avance II 600 instruments (Bruker, Billerica, MA, USA). NMR spectra recorded in
CDCl3 were referenced to the respective residual 1H or 13C signals of the solvent; chemical shifts are
expressed in ppm, and coupling constants in Hz. Infrared spectra (4000–400 cm−1) were collected
on a Perkin Elmer 2000 FTIR spectrometer (PerkinElmer, Waltham, MA, USA). High-resolution mass
spectra were collected using electrospray ionization on a Waters LCT Premier XE TOF instrument
(Waters Corporation, Milford, MA, USA). Optical rotations were measured using an Model AA-5
automatic polarimeter (Optical Activity, Ltd., Ramsey, UK); [
−1. Chromatographic separations were performed on silica gel 60 (70–230 mesh). Thin-layer
α]
D
values are given in 10−1 deg cm2
g
chromatography was carried out using silica gel 60 precoated plates.
X-ray diffraction data for monocrystals were collected on an Xcalibur Ruby diffractometer [23
]
with MoKα radiation at 80(2) K. The Cambridge Crystallographic Data Centre (CCDC) reference
numbers for 12a and 12d: CCDC 1992131 (12a) and 1992390 (12d) (Supplementary data available from
CCDC, 12 Union Road, Cambridge CB2, 1EZ, UK on request). The crystal structures were solved
with direct methods in SHELXT and refined using the full-matrix method in SHELXL [24]. H atoms
bonded to C atoms were generated using HFIX instruction with Ueq = 1.2–1.5Ueq (parent C atom).
Basic crystallographic data are presented in Supplementary Table S1.
2.2. Preparation of Starting Compounds
The bicyclic amines, (1S,3R,4R)-2-[(S)-1-phenylethyl]-3-aminemethyl-2- azabicyclo[2.2.1]heptane
and (1S,4S,5R)-2-[(S)-1-phenylethyl]-4-amine-2- azabicyclo[3.2.1]octane , were prepared as described
previously [20 21]. The synthesis of enantiopure 2-azanorbornane amine derivatives containing two
bicyclic units 8 and 9 was also reported by our group [21].
2
3
,

Molecules 2020, 25, 2355
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2.3. General Procedure for Sulfonamide Synthesis
The appropriate sulfonyl chloride (1.0 mmol) was added to a solution of primary amine 2 or 3
(0.23 g, 1.0 mmol) and powdered potassium hydroxide (0.10 g, 1.8 mmol) in a dry dichloromethane
(15.0 mL), which was stirred at room temperature. After 24 h, the resulting reaction mixture was poured
over brine and extracted with dichloromethane. The organic phases were dried over Na2SO4, filtered
through the Celite®, washed with dichloromethane, and evaporated under vacuum. The resulting
residue was purified by column chromatography on silica (eluent: ethyl acetate/n-hexane 1:1 v/v),
yielding sulfonamide 10 (from amine
to the synthesis of compound 13a from amine
(0.25 g, 1.0 mmol); the n-hexane/ethyl acetate (3/1 v/v) mixture was used for elution of the product.
For synthesis of compounds bearing two sulfonamide functions, 12, bisamine (0.48 g, 1.0 mmol)
2
) or 11 (from amine
3). An analogous procedure was applied
9
(0.44 g, 1.0 mmol) and biphenyl-4-sulfonyl chloride
8
was reacted with 2.0 mmol of the appropriate sulfonyl chloride; final products were eluted with
n-hexane/ethyl acetate (3/1 v/v for 12a, 1/1 v/v for the remaining derivatives). The physicochemical
characterization of all newly obtained compounds (including copies of 1H and 13C NMR spectra, and
X-ray structures of compounds 12a and 12d) can be found in the Supplementary Materials.
2.4. Cell Lines and Culture Cconditions
The human hepatocellular carcinoma (HCC) cell line HUH7 was purchased from ATCC (LGC
standards GmbH, Wesel, Germany). The Austrian human HCC cell line AKH12 has been well
characterized [21–25] and was kindly provided by Prof. Bettina Grasl-Kraupp. HUH7 cells were
cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA)
containing 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA),
1% penicillin-streptomycin (10,000 U/mL) (Thermo Fisher Scientific, Waltham, MA, USA), and 1%
minimum essential medium nonessential amino acids (100x) (Thermo Fisher Scientific, Waltham,
MA, USA). AKH12 cells were cultured in RPMI1640 medium (Sigma-Aldrich, St. Louis, MO, USA)
containing 10% heat-inactivated FBS. DAOY and D283Med (short D283) medulloblastoma (MB) cells
were purchased from ATCC (LGC standards GmbH, Wesel, Germany). D425Med (short D425) and
UW228-2 were provided by Thomas Ströbel from the Institute of Neurology Medical University of
Vienna. The cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis,
MO, USA) containing 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), 2% l-Glutamine (Sigma-Aldrich,
St. Louis, MO, USA), and 0.2% Normocin (Sigma-Aldrich, St. Louis, MO, USA). U-251 MG (short
U251) glioblastoma (GBM) cell line was purchased from Sigma-Aldrich and cultured in Dulbecco’s
modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Sigma-Aldrich,
St. Louis, MO, USA), 2% l-Glutamine (Sigma-Aldrich, St. Louis, MO, USA), and 0.2% Normocin
(Sigma-Aldrich, St. Louis, Missouri, USA). Human umbilical vein endothelial cells (HUVECs) were
provided by Brigitte Winter from the Department of Surgery Medical University of Vienna and were
grown in endothelial cell growth medium-2 BulletKitTM (Lonza, Basel, Switzerland) under standard
tissue culture conditions. All cells were incubated at 37 ◦C in a 5% CO2 atmosphere and regularly
checked for mycoplasma contamination.
2.5. Cell Viability Assay (Adherent Cell Lines)
Cell viability was determined using a neutral red assay, as previously described [26]. The cells
were placed in 24-well plates and grown to reach 70–80% confluency prior to treatment. After treatment
for 24 h, cells were incubated with neutral red at a concentration of 50 µg/mL in serum-free medium
for 2 h at 37 ◦C. Cells were then washed 2x and incubated with 1% acetic acid in 70% ethanol. The dye
concentration was measured photometrically at 562 nm. The percentage of survival was calculated
considering DMSO-treated (indicated as 0
µM concentration) cells as 100% control. The final DMSO
concentration in media did not exceed 0.2%.

Molecules 2020, 25, 2355
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LD50 was defined as the amount of a toxic agent required to eliminate 50% of the cell population.
The LD50 was calculated in each experiment from the respective dose–response curve. All experiments
were performed in triplicate wells.
2.6. Cell Viability Assay (Suspension Cell Line)
One day before treatment, 2
×
104 of D425Med or HUVEC cells were seeded into 24-well plates
and incubated at 37 ◦C in a humidified atmosphere containing 5% CO2.The cells were treated with
different compounds for 24 h. A MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay using the CellTiter Blue reagent (Promega, Fitchburg, Wisconsin, USA) was performed according
to the manufacturer’s protocol. Experiments were performed in triplicate and repeated three times.
The percentage of survival was calculated as the ratio using DMSO-treated (indicated as 0
µM
concentration) cells as 100% control. The final DMSO concentration in media did not exceed 0.2%.
2.7. Determination of Lactate Dehydrogenase (LDH)
LDH release into culture media was measured by a cytotoxicity detection kit from Roche
Diagnostics, according to the manufacturer’s instruction. LDH-positive controls or 100% lysed cells
were generated in each cellular experiment by addition of Triton X100. Additionally, 0.2% DMSO was
used as the vehicle control.
3. Results and Discussion
3.1. Preparation of Compounds
Enantiopure isomeric amines based on bicyclic skeletons of 2-azabicyclo[2.2.1]heptane 2 and
2-azabicyclo[3.2.1]octane
azabicyclo[2.2.1]heptane
3
were prepared from (1S,3R,4R)-2-[(S)-1-phenylethyl]-3- hydroxymethyl-2-
4, as previously described [19 21]. Nucleophilic substitution of the hydroxyl
,
group of alcohol
, which was then reduced with triphenylphosphine to yield amine
containing a preserved bicyclic system, was obtained using a three-step route involving Swern oxidation
of alcohol , conversion of the resulting aldehyde to oxime , and its reduction. Amines containing two
bicyclic fragments and were also reported in our previous paper [21]; their preparation included
reaction of aldehyde with ethylenediamine or amine , respectively, followed by reduction of the
obtained imines with sodium borohydride (Scheme 1).
4
with azide resulted in ring expansion, exclusively giving one stereoisomeric product
7
3
(Scheme 1). In turn, amine 2,
4
6
8
9
5
2
Scheme 1. Synthesis of amines 2, 3, 8, and 9.
Amines
2,
3
,
8, and
9
were converted into a series of sulfonamides. To this end, they were
reacted with various sulfonyl chlorides in dichloromethane solution in the presence of powdered
potassium hydroxide (Scheme 2). After chromatographic purification, they were obtained as crystalline
solids or oils at 30–85% yield. Their structures were confirmed by spectroscopic methods (FTIR, 1H,
and 13C NMR) and high-resolution mass spectrometry (data available in Supplementary Materials).

Molecules 2020, 25, 2355
5 of 10
In this way, enantiomerically pure derivatives 10 and 11, differing in the size of the bicyclic skeleton
and the distance of sulfonamide function from the chiral scaffold, were obtained together with
compounds possessing two 2-azanorbornane subunits and one (13) or two (12) sulfonamide fragments.
The introduced substituents included biphenyl (10a-13a), substituted biphenyl (10b-10e
12d), trifluoromethylated phenyls (10f-g 11f-g), chiral camphoryl moiety (10h 11h), and acetamide
derivative (12i 13i). The choice of these groups was substantiated by their presence in various drugs
, 11e, 12b, and
,
,
,
and therapeutics. Besides full spectroscopic characterization, structures of derivatives 12a and 12d
were additionally confirmed by X-ray crystallography (structures shown in Supplementary Materials).
We further investigated the impact of the sulfonamide structure on their cytotoxicity.
Scheme 2. Synthesis of sulfonamides 10–13.
3.2. Primary Evaluation of the Anticancer Activity of Sulfonamide Compounds
All tested compounds were dissolved in dimethyl sulfoxide (DMSO). The solubility of compounds
12b
,
12d
,
8
, and
9
was not sufficient for our in vitro study. In the first screening, compounds that reduced
M after 24 h were considered as positive hints and were eligible
cell viability by more than 50% at 50
µ

Molecules 2020, 25, 2355
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for lactate dehydrogenase (LDH) cytotoxicity assay. To assess the anticancer activity of compounds,
we tested human hepatocellular carcinoma (HCC), medulloblastoma (MB), and glioblastoma (GBM)
cell lines. Further, to compare the effects of selected compounds on nonmalignant primary cells, we
used primary human umbilical vein endothelial cells (HUVEC).
3.3. Sulfonamides and HCC Cell Lines
HCC accounts for a vast majority of liver cancer and is among the leading causes of cancer-related
mortality [27]. HCC has been considered as a highly chemoresistant cancer, and hence is a good
candidate for our study. The introduced 50
11g 12i, and 13a compounds reduced cell viability below LD50 in HUH7 and AKH12 cell lines (Table 1;
Figures S1A and S3A available in Supplementary Materials). 50 M concentration of 11f, however,
µM concentration of 10a, 10b, 10c, 10e, 10f, 10g, 11a, 11e,
,
µ
only diminishes viability of HUH7 cells by more than 50%. The cytotoxicity of mentioned compounds
was measured by the LDH release in a dose-dependent manner (Supplementary Figures S1B and S3B).
The cytotoxicity was compared between HCC and HUVEC cells. Our results showed no significant
differences of sulfonamide cytotoxicity in HCC cell as compared to HUVEC cells (Supplementary
Figures S2 and S4—data available in Supplementary Materials). LD50 values for the above compounds
tested in HUH7, AKH12, and HUVEC cells are summarized in Table 1.
Table 1. LD50 values for specified sulfonamides tested in Huh7, AKH12, DAOY, UW228-2, D283 and
U251 tumor cell lines, as well as in human umbilical vein endothelial cells (HUVEC) cells.
Cell Line/LD50 [µM]
HCC
MB
GBM
U251
Compound
HUVEC
HUH7
AKH12
DAOY
UW228-2
D283
10a
10b
10c
10e
10f
10g
11a
11e
11f
11g
12i
13.94 ± 1.02
21.33 ± 1.23
17.33 ±0.98
16.33 ± 0.69
24.57 ± 1.54
11.66 ± 1.53
22.5 ± 1.21
30.22 ± 2.21
42.66 ± 1.98
20 ± 1.1
15.5 ± 1.3
23.72 ± 1.27
23.27 ± 2.1
32 ± 2.21
24.72 ± 1.98
10.18 ± 1.09
28.8 ± 1.53
28 ± 1.39
-
16 ± 1.12
28.8 ± 2.1
17.71 ± 1.09
31.27 ± 1.32
27.25 ± 2.35
15.61 ± 0.59
18.78 ± 1.22
16 ± 1
13.51 ± 0.9
20.44 ± 1.58
21.33 ± 1.01
16 ± 2
11.58 ± 1.11
12.88 ± 2.13
13.8 ± 1.14
9.58 ± 1.88
17.26 ± 2.33
10.77 ± 0.93
13.35 ± 0.25
17.58 ± 2.94
22.28 ± 1.45
18.68 ± 1.97
3.16 ± 0.15
12.13 ± 0.26
-
-
16 ± 2.1
15.74 ± 1.32
26.66 ± 1.52
28 ± 2.09
15.54 ± 1.62
15.77 ± 1.09
19.2 ± 1.42
18.66 ± 1.88
24 ± 1.49
32.72 ± 1.32
11.76 ± 1.58
18.78 ± 2.11
23.7 ± 1.69
31.48 ± 2
12 ± 0.99
16 ± 1.03
20 ± 2.02
23.46 ± 1.56
31.36 ± 2.12
20.14 ± 1.34
28.44 ± 2.11
25.9 ± 1.98
25.9 ± 1.9
17.33 ± 0.99
20.41 ± 1.33
12.88 ± 1.59
19.2 ± 1.37
29.91 ± 1.5
17.23 ± 0.89
21.81 ± 1.9
38.4 ± 1.11
28 ± 1.72
5.75 ± 0.47
8 ± 0.28
24.8 ± 0.92
15.33 ± 1.59
27.63 ± 1.04
7.75 ± 0.84
13.92 ± 0.49
13a
3.4. Sulfonamides and MB Cell Lines
MB is considered the most common malignant brain tumor, with an estimated 5000–8000 cases per
year worldwide [28]. Current MB classification defines four molecular subgroups, namely wingless
(WNT), sonic hedgehog (SHH), group 3, and group 4 [29]. Among the MB cells tested, DAOY and
UW228-2 belong to the SHH subgroup, whereas D425 corresponds to group 3. Cell line D283 exhibits
features of both group 3 and group 4 [30]. Here, 50
11f 11g 12i, and 13a reduced cell viability in all 4 cell lines (Supplementary Figures S5A, S7A, S9A
and S11A—data available in Supplementary Materials). Compound 10b, however, only affected D425
and D283 cells, with LD50 values of 16.38 2.34 M and 31.27 1.32 M, respectively (Tables 1 and 2).
LDH release indicative for loss of membrane integrity is shown in Supplementary Figures S5B, S7B,
S9B, and S11B. Among the compounds tested, 10b 10c 10e 10g, and 11e significantly reduced D425
cell viability as compared to HUVEC cells (* p 0.05, and ** p 0.01; Supplementary Figure S10—data
available in Supplementary Materials). The LD50 values for the designated sulfonamides in all MB
cell lines and HUVEC cells are shown in Tables 1 and 2. LD50 values for compounds 10c (5.38 M)
and 10g (6.61 M) in D425 cells (Table 2) were slightly below the LD50 values of the above known
µM of compounds 10a, 10c, 10e, 10f, 10g, 11a, 11e,
,
,
±
µ
±
µ
,
,
,
≤
≤
µ
µ
chemotherapeutics, etoposide, and cisplatin (8 and 10
Bueren et al. [31].
µM, respectively) reported in the study by von

Molecules 2020, 25, 2355
7 of 10
Table 2. LD50 values for designated sulfonamides tested in D425 and HUVEC cells.
Cell line/LD50 [µM]
MB
D425
HUVEC/D425 LD50
Compound
HUVEC
Fold Change
10a
10b
10c
10e
10f
10g
11a
11e
11f
11g
12i
16.71 ± 2.11
16.38 ± 2.34
5.38 ± 0.77
10.95 ± 0.47
20.24 ± 0.79
6.61 ± 1.52
21.42 ± 0.89
17.78 ± 1.04
32 ± 1.37
28 ± 1.86
28.9 ± 2.97
16.77 ± 0.71
11.54 ± 2.8
23.15 ± 2.21
17.62 ± 2.98
21.71 ± 1.99
36.38 ± 4.37
31.52 ± 1.16
16 ± 2.29
1.67
1.76
3.11
1.05
1.14
2.66
1.01
2.04
0.98
1.42
0.57
0.52
11.2 ± 1.73
15.6 ± 0.93
30.54 ± 1.86
8.91 ± 2.11
16 ± 0.79
13a
3.5. Sulfonamides and GBM Cell Line
Despite aggressive therapy, GBM continues to be associated with a dismal prognosis in virtually
all adult patients [31]. U251, a GBM cell line, was subjected to 50 M of sulfonamides. Compounds
10a 10b 10c 10e 10f 10g 11a 11e 11f 11g 12i, and 13a decreased cell viability by more than
µ
,
,
,
,
,
,
,
,
,
,
50% (Supplementary Figure S13A—data available in Supplementary Materials). The LDH (lactate
dehydrogenase) cytotoxicity assay was performed to assess necrotic cell death (Supplementary Figure
S13B—data available in Supplementary Materials). There were no statistically significant differences
in cytotoxicity between U251 cells as compared to HUVEC cells (Supplementary Figure S14—data
available in Supplementary Materials). Table 1 represents the LD50 values of the tested sulfonamide on
U251 and HUVEC cell lines.
3.6. SAR Analysis
In our previous investigations, amines derived from 2-azanorbornane were prepared and tested
for antiproliferative activity using three cancer cell lines: human lung cancer A549, human colon
cancer HT-29, and biphenotypic leukemia MV4-11 [21]. The selectivity was assessed with human
lung microvascular endothelial cells (HLMEC) and BALB/3T3 mouse embryo fibroblasts. Bisamine
compounds 8 and 9, two compounds that can be treated as reference for the current study, exhibited
rather low cytotoxicity, although for compound 8, the IC50 value for colon cancer line was comparable
to cisplatin (7.2
(IC50 values for AT549, HT-29, and MV4-11 equaled 4.1
respectively). However, it also inhibited the growth of mouse fibroblasts (IC50 =19.7
nonmalignant endothelial cells (IC50 = 5.8 ± 0.6 µM), meaning its selectivity was unsatisfactory.
We planned to use compounds and as references in the current study. Unfortunately, they were
±
0.8
µM vs 9.7
±
2.0
µ
M). The N-Benzyl derivative of compound 9 was more active
0.1 M, 4.8 0.4 M, and 5.4 0.7 M,
10.1 M) and
±
µ
±
µ
±
µ
±
µ
8
9
found to be insufficiently soluble in the DMSO used for the tests. In contrast, most of the sulfonamide
derivatives showed better solubility, and we could perform all tests with their samples. In our further
studies, we plan to compare activities of all compounds using ethanol as the solvent.
Although the activities of the investigated sulfonamides varied from one cell line to another, certain
regularities could be observed, which are important from the point of view of further developments.
Comparison of activities exhibited by isomeric compounds 10 and 11, containing bicyclic systems of
various sizes, showed that 2-azanorbornyl derivatives (in particular, 10b, 10c, 10e, and 10g) exhibited
higher cytotoxicity than their 2-azabicyclo[3.2.1.]octane (bridged azepane) counterparts. The scaffold
of the latter can be regarded as less rigid; on the other hand, higher flexibility of the arm containing
sulfonamide moiety in 2-azabicylo[2.2.1]heptane-derived compound 10 seems to be of importance as
well. Among substituents present in the sulfonamide, biphenyl-containing methoxy group (10e, 11e)

Molecules 2020, 25, 2355
8 of 10
or fluorine (10c) were found to be most active. For phenyl derivatives, the introduction of two –CF3
moieties resulted in enhanced activity and selectivity (compounds 10g 11g), while one trifluoromethyl
,
group in the para position was not sufficient to increase the antiproliferative properties.
Finally, dimeric species 12i and 13a were not particularly active, and even their toxicity for normal
cell lines was higher than for malignant ones.
4. Conclusions
In this study, we evaluate the effect of chiral 2-azabicycloalkane-based sulfonamides on a variety
of tumor cell lines, including HCC, MB, and GBM. Our results indicate that compounds 10b
, 10c, 10e,
10g, and 11e notably reduce cell viability on the D425 cell line (* p 0.05, and ** p 0.01) as compared
≤
≤
to HUVEC cells. The D425 cell line is categorized as a group 3 medulloblastoma, with high MYC
proto-oncogene amplification [29]. In D425, the best discrimination in cytotoxicity between malignant
and nonmalignant cells was observed for compound 10c (3.11-fold change), followed by compounds
10g and 11e (2.66- and 2.04-fold changes, respectively). Thus, compounds 10c, 10g, and 11e showed
the highest performance among the agents tested in this study. Although the safety margin seems to
be too narrow and remains far below the >30-fold difference recommended for safety reasons [32],
appropriate modifications can be introduced to optimize the structures of the studied sulfonamides.
These promising stable compounds can serve as a convenient starting point for further exploration.
Supplementary Materials: The following are available online. Figure S1: Effect of sulfonamide compounds
on HUH7 cell line, Figure S2: Impact of sulfonamide compounds on HUH7 in contrast to HUVEC, Figure S3:
Viability and cytotoxicity effect of sulfonamide compounds on AKH12 cells, Figure S4: Impact of sulfonamide
compounds on AKH12 compared to HUVEC, Figure S5: Influence of sulfonamide compounds treatment on DAOY
cell viability and cytotoxicity, Figure S6: Sulfonamide compounds sensitivity of DAOY in contrast to HUVEC
cell lines, Figure S7: Effect of sulfonamides on viability of UW228-2 cell line, Figure S8: Impact of sulfonamides
on UW228-2 in contrast to HUVEC, Figure S9: Sulfonamides sensitivity and cytotoxicity on D425 cells, Figure
S10: Effect of sulfonamides on viability of D425 in comparison with HUVEC cell lines, Figure S11: Influence of
sulfonamide compounds treatment on D283 cell viability and cytotoxicity, Figure S12: Efficacy of sulfonamides on
cell viability of D283 compared to HEVEC cell lines, Figure S13: Sulfonamides and viability of U251 cells, Figure
S14: Efficacy of sulfonamides on cell viability of U251 compared to HEVEC cell lines, Figure S15: X-Ray structure
of bis-sulfonamide 12a, Figure S16: X-Ray structure of bis-sulfonamide 12d, Table S1: X-Ray data for compounds
12a and 12d; spectral data for new compounds and copies of 1H NMR and 13C NMR spectra.
Author Contributions: Conceptualization, N.R.-U. and E.W.; data curation, M.S., M.S., D.S., S.M., D.I., K.K.,
J.W., and A.K.; investigation, M.S., M.P., D.S., S.M., D.I., K.K., J.W., and A.K.; supervision, N.R.-U. and E.W.;
writing—original draft, N.R.-U., M.S., and E.W.; writing—review and editing, D.I., K.K., and J.W. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors thank Kerstin Zinober and Martha Seif for their support in cell culture experiments.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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