Taurultams incorporating arylsulfonamide: First in vitro inhibition studies of α-, β- and γ-class Carbonic Anhydrases from Vibrio cholerae and Burkholderia pseudomallei

Article abstract of DOI:10.1016/j.ejmech.2021.113444

A new series of taurultambenzenesulfonamides 1–17 were prepared and considered for their inhibitory activity in vitro against the Carbonic Anhydrases from Vibrio cholerae (VchCA-α, VchCA-β and VchCA-γ) and Burkholderia pseudomallei (BpsCA-β and BpsCA-γ). Among the compounds tested, derivatives 4, 5, 7, 10, 12, and 16 resulted in highly effective VchCAα inhibitors (K_I values spanning within the 6.1–9.6 nM range) and endowed with excellent Selectivity Indexes (SIs; K_I VchCA-α/K_I hCA II) all comprised between 0.04 and 0.09. Potent in vitro inhibitors for the BpsCA-γ were also identified (K_Is of 18.9–19.5 nM). The results here reported may represent the blueprint for the future development of a new generation of CA-based antibiotics integrated with free of resistance mechanisms of action adopted from known drugs.

Full text of DOI:10.1016/j.ejmech.2021.113444

European Journal of Medicinal Chemistry 219 (2021) 113444
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Taurultams incorporating arylsulfonamide: First in vitro inhibition
studies of
a-, b- and g-class Carbonic Anhydrases from Vibrio cholerae
and Burkholderia pseudomallei
,
, c,
Ozlem Akgul a *, Andrea Angeli b **, Silvia Selleri b, Clemente Capasso d,
Claudiu T. Supuran b, Fabrizio Carta b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ege University, 35100, Bornova, Izmir, Turkey
a
_
b
ꢀ
Universita degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019, Sesto Fiorentino
(Florence), Italy
c Centre of Advanced Research in Bionanoconjugates and Biopolymers - "Petru Poni", Institute of Macromolecular Chemistry, 700487, Iasi, Romania
d Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of taurultambenzenesulfonamides 1e17 were prepared and considered for their inhibitory
Received 13 December 2020
Received in revised form
27 March 2021
Accepted 30 March 2021
Available online 15 April 2021
activity in vitro against the Carbonic Anhydrases from Vibrio cholerae (VchCA-
and Burkholderia pseudomallei (BpsCA- and BpsCA- ). Among the compounds tested, derivatives 4, 5, 7,
10, 12, and 16 resulted in highly effective VchCA inhibitors (KI values spanning within the 6.1e9.6 nM
range) and endowed with excellent Selectivity Indexes (SIs; KI VchCA- /KI hCA II) all comprised between
0.04 and 0.09. Potent in vitro inhibitors for the BpsCA- were also identified (KIs of 18.9e19.5 nM). The
a, VchCA-b and VchCA-g)
b
g
a
a
g
results here reported may represent the blueprint for the future development of a new generation of CA-
based antibiotics integrated with free of resistance mechanisms of action adopted from known drugs.
© 2021 Elsevier Masson SAS. All rights reserved.
Keywords:
Carbonic anhydrase inhibitors (CAIs)
Benzensulfonamide
Taurultam
Taurine
Vibrio cholerae
Burkholderia pseudomallei
1. Introduction
food security, agriculture, breeding, biosafety and social develop-
ment [2]. In such a scenario, bacterial expressed Carbonic Anhy-
The recent metadata analysis on antimicrobial resistances
(AMRs) at global scale ascribed at least 700.000 death per year to
bacterial resistances, with a prediction up to 10 million by 2050 if
no effective preventive measures control, control and development
of the phenomena are considered [1]. Misuse of antibiotics is un-
doubtedly the primary cause of bacterial resistance as it dramati-
cally accelerates the natural survival process of bacteria when
exposed to xenobiotics [1]. The problem is of particular concern for
the World Health Organization (WHO), as it significantly reinforced
antibiotic resistance surveillance and action programs in sanitary,
drases (CAs, EC 4.2.1.1) are of particular interest since i)
CAs are encoded and expressed only from bacteria. As a side note
a
-,
b
- and
g
a
-
-
CAs in prokaryotes are structurally different from their human
expressed homologues [3]; ii) they retain the essential role in
sustaining and regulating microorganism’s metabolism and viru-
lence by catalyzing the carbon dioxide hydration equilibrium [3].
Such a minimal transformation is of paramount importance among
all living organisms. It is the only way to modulate timely any
addition/detraction of carbon into/from its biologically fruitful form
(i.e., CO2/HCOꢀ3 respectively) [3]. It is therefore expected that any
disruption of the CA activity will heavily affect the microorganism’s
biology. Such an idea is rapidly thriving and gaining consensus
among the scientific community [4e8]. Although the work here
presented lies on the same principle, it contains as innovative
feature the molecular hybridization technique applied to the broad
spectrum antiinfective Taurolidine 1. Such a molecule exerts anti-
bacterial activity by means of its hydrolysis/decomposition
* Corresponding author.
ꢀ
** Corresponding author. Universita degli Studi di Firenze, NEUROFARBA Dept.,
Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019, Sesto
Fiorentino (Florence), Italy.
E-mail addresses: ozlem.akgul@ege.edu.tr (O. Akgul), andrea.angeli@unifi.it
(A. Angeli).
https://doi.org/10.1016/j.ejmech.2021.113444
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

O. Akgul, A. Angeli, S. Selleri et al.
European Journal of Medicinal Chemistry 219 (2021) 113444
products hydroxymethyl taurultam 2 and methylol taurinamide 4,
which bind irreversibly to the cell-wall lipopolysaccharides con-
stituents, thus determining inhibition of bacterial adhesion to host
cells and lysis (Scheme 1) [9].
derivatives. The structures of all final compounds and in-
termediates were determined through 1H NMR, 13C NMR and HRMS
analysis.
The high safety profile and scarce bacterial resistances of Taur-
2.2. Carbonic anhydrase inhibition
olidine
1 prompted us to investigate merging of its 1,2,4-
thiadiazinane-1,1-dioxide core into CA inhibitors (CAIs) of the sul-
fonamide type and to profile them in vitro against CAs expressed
from bacterial strains of particular concern such as the Gram-
All the newly prepared taurultamsulfonamide bearing com-
pounds 1e17 were screened in vitro for their potential CA inhibitory
features against the VchCA-a, VchCA-b, VchCA-g, BpsCA-b, BpsCA-g
negative Vibrio cholerae (i.e., VchCA-
Burkholderia pseudomallei (i.e., BpsCA-
a
, VchCA-
b
and VchCA-
) [3,7].
g) and
and compared to the off-target human (h) expressed CAs I and II
tested under the same conditions. The experiments were per-
formed by means of stopped-flow CO2 hydrase assay [25] using the
standard sulfonamide inhibitor acetazolamide (AAZ) and sulthiame
(SLT) as reference drugs. The obtained data are reported in Table 1.
Based on data in Table 1, the following structure-activity re-
lationships (SARs) can be drawn:
b
and BpsCA-
g
V. cholerae usually is transmitted to humans through contami-
nated water and/or food and is the etiological agent of cholera
disease, which determines up to 91.000 deaths annually and
reached the pandemic level 7 times [10,11]. Various resistances to
antibiotics were reported for V. cholerae with consequent failure of
pharmacologically applied protocols [11].
B. pseudomallei are the causative agent of melioidosis, also
referred to as Whitmore’s disease or pseudoglanders. The pathogen
can be acquired from environmental sources by either inhalation,
ingestion or inoculation and may lead to chronic or acute infections
[12,13]. B. pseudomallei can reactivate years after pharmacological
treatment. Since it has the potential to be measured as a biological
threat, the Centers for Disease Control (CDC) classified such a
ꢁ The VchCA-
a from V. cholerae was inhibited by 1e17 with KI
values spanning within the range of 6.1 and 894.4 nM. Notably,
the substitutions at 4-position of the phenyl distal tail proved
very sensitive in varying the inhibitory kinetic profiles on such
isoform. For instance, within the sulfanilamide series, the
introduction in 1 of a methyl, fluoro and methoxy, as in 2, 3 and
8, raised the KI inhibition values to 592.9 nM. Consistent varia-
tions of the inhibition potencies for 1e5 were obtained when
the sulfanilamide warhead was elongated by means of the
introduction of the ethyl spacer as in 10e14. For instance, the
inhibition potency of the unsubstituted compound 1 and the 4-
fluoro derivative 3 to afford 10 and 12 resulted increased up to
10.5- and 82-fold, respectively. Conversely, the same chemical
transformation resulted detrimental for the inhibition potency
for 2, 4 and 5 as their corresponding longer derivatives showed
KI values risen up to 1.78-, 84.4- and 10.5-fold, respectively (see
Table 1). Among the remaining compounds tested against the
pathogen as
a Tier 1 Select agent [14]. Several strains of
B. pseudomallei were reported resistant to common antibiotics (i.e.,
penicillin, ampicillin, first- and second-generation cephalosporins,
gentamicin, tobramycin, streptomycin, macrolides and polymyxins)
[12e14].
2. Results and discussion
2.1. Design and synthesis of compounds
For this project, we sought to insert the 1,2,4-thiadiazinane 1,1-
dioxide moiety as a linker interconnecting the benzene sulfon-
amide CAI warhead with the distal section of the molecule as
schematically reported in Fig. 1.
In agreement with the design strategy in Fig. 1, we prepared a
series of compounds 1e17 by using a 6 step reaction procedure
(Scheme 2) and partially reported by some of us [15,16].
The obtained key intermediate 2-phthalimidoethanesulfonyl
chloride A [15,16] was reacted with the appropriate commercially
available arylsulfonamide to furnish 4-((2-(1,3-dioxoisoindolin-2-
yl)ethyl)sulfonamido)benzenesulfonamide B1 and 4-(2-((2-(1,3-
dioxoisoindolin-2-yl)ethyl)sulfonamido)ethyl)benzenesulfona-
mide B2 [17,18], followed by removal of the phthalimido protecting
group [19e21]. The tail section was installed by treatment of the in
situ released primary amine with various benzyl chlorides at r.t. to
afford derivatives D1-D17 [22,23]. Then the 1,2,4-thiadiazinane-1,1-
dioxide was installed by cyclization of with 37% formaldehyde in
methanol at pH ¼ 13 under ultrasound irradiation for 8 h [24] to
afford the 4-((substitutedbenzyl)-1,1-dioxido-1,2,4-thiadiazinan-2-
yl)benzenesulfonamide 1e9 and 4-(2-((substitutedbenzyl)-1,1-
dioxido-1,2,4-thiadiazinan-2-yl)-ethyl)benzenesulfonamide 10e17
VchCA-a of note are the perfluorophenyl 4 and the 2,6-
difluorophenyl 16 with KIs of 8.5 and 8.0 nM, which are close
to the reference AAZ (KI of 6.8 nM).
ꢁ Overall, the VchCA-
b was poorly inhibited by the series of
compounds tested compared to the previously discussed
a-
class. The kinetic data showed that the introduction at position 4
within the unsubstituted phenyl tail on 1 of an electron-
donating moiety either of small or large dimensions, as in 2, 6
and 8, determined a slight increase of the binding potency. The
KIs of the obtained compounds were 343.0, 349.2 and 344.2 nM,
thus lower than the reference AAZ (KI 451.0 nM). In this case,
elongation of the CAIs benzenesulfonamide connection heavily
potentiated their inhibition potency only of the electron with-
drawing containing moieties such as 3e5 to afford 12e14, with
the latter being 19.5-, 96.1- and 74.3-fold respectively more
potent. Among the remaining compounds, which resulted in all
micromolar VchCA-
is worth of consideration as it showed a KI value of 211.9 nM.
when incubated to CAIs 1e17
b inhibitors, only the 4-chloro derivative 17
ꢁ The kinetic profile of the VchCA-
g
lacks any significative SAR as all the values are in the micromolar
range with tiny differences among them. The most potent
Scheme 1. Hydrolysis and decomposition of Taurolidine 1 to its active fragments Hydroxymethyl taurultam 2 and Methylol Taurinamide 4 [9].
2

O. Akgul, A. Angeli, S. Selleri et al.
European Journal of Medicinal Chemistry 219 (2021) 113444
Fig. 1. Schematic representation of the design strategy adopted.
Scheme 2. Synthesis of taurultam benzenesulfonamides 1e17.
compound resulted in 4-fluorosubstituted derivative 4 with a KI
value of 6400 nM, thus 13.5-fold less potent then the reference
AAZ (KI of 473 nM) (see Table 1).
the inhibition potencies (KIs of 953.4 and 823.6 nM respec-
tively), which in turn were partially restored when the bulky 4-
isopropyl and the 3-chloro groups were inserted (KIs of 42.9 and
68.7 nM). Among the longer series 10e17, only the 4-nitro 14
and the 2,6-difluoro 16 derivatives showed better inhibition
potencies when compared to the unsubstituted compound 10
(KIs of 18.9, 19.5 and 45.3 nM respectively), whereas the
remaining ones gave KI values spanning between 105.8 and
502.8 nM.
ꢁ As for the BpsCA-b, the compounds tested resulted in high nano/
low micromolar inhibitors with only few exceptions accounting
for low nanomolar KIs such as the derivatives 7, 10 and 17 (KIs of
81.8, 83.0, 65.8 nM respectively). A rather undefined SAR is
obtained from the data reported in Table 1. Of consideration are
the variation of the KI values of compounds when elongation of
the benzenesulfonamide warhead was accomplished. For
instance, the unsubstituted phenyl derivatives 10, 12 and 13
resulted in 7.9-, 19.3- and 2.5-fold more potent than their
shorter counterparts 1, 3 and 4.
To explore any preferential inhibition of our compounds among
the bacterial and human expressed CAs, we also considered the
in vitro inhibition against the hCAs I and II. The results are pre-
sented in Table 1.
ꢁ The second expressed BpsCA class (i.e. the
g) resulted more
effectively inhibited from the compounds tested. The unsub-
stituted derivative 1 was an effective inhibitor with a KI of
37.0 nM. It was up to 4.0-fold more effective when compared to
the reference AAZ (KI of 149 nM). Interestingly, the introduction
in 1 of the fluorine atom as in compound 3 determined
enhancement of the KI value up to 67.6 nM, whereas the tri-
fluoromethyl derivative 4 resulted particularly potent with a KI
of 19.1 nM. Conversely, the introduction of the nitro or penta-
fluorophenyl electron withdrawing groups, as in 5 and 7, spoiled
ꢁ The sulfanilamide (1e9) and ethylsulfanilamide (10e17) de-
rivatives were high nanomolar inhibitors for hCA I and hCA II,
with KI spanning between 941.7-7863 nM and 629.1e7740 nM,
respectively. The best hCA I inhibitors resulted in the fluoro
substituted compound (3), the phenyl (10), the tolyl (11) and
trifluoromethyl (13) derivatives with KI values ranging between
629.1 and 941.7 nM (see Table 1)
3

O. Akgul, A. Angeli, S. Selleri et al.
European Journal of Medicinal Chemistry 219 (2021) 113444
Table 1
Stopped-flow kinetic data of compounds 1e17, AAZ and SLT on VchCA-
a
, VchCA-
b, VchCA-
g
, BpsCA-
b
, BpsCA-
g
and hCAs I, II.
KI (nM)a
Cmp
R
VchCA-
a
VchCA-
b
VchCA-
g
BpsCA-
b
BpsCA-
g
hCA I
hCA II
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
-H
4-CH3
4-F
4-CF3
4-NO2
4-CH(CH3)2
1,2,3,4,5-F
4-OCH3
3-Cl
-H
4-CH3
4-F
4-CF3
4-NO2
4-C(CH3)3
2,6-F
76.4
502.7
500.0
9.6
8.9
81.4
8.5
592.9
37.7
7.3
894.4
6.1
810.7
94.0
96.2
8.0
678.5
451
432
343
7300
6542
6400
8920
7164
9160
8109
7686
6771
8620
8524
8235
8666
8575
8882
8815
8888
745
656.3
601.7
4263
609.7
437.5
4649
81.8
410
415.8
83.0
6173
221.3
248.3
5500
276.5
393.2
65.8
37
116.9
67.6
19.1
953.4
42.9
823.6
705.8
68.7
4652
5430
941.7
7641
7863
4207
2441
4489
3410
763.5
629.1
1905
647.8
4413
7740
5378
2054
12.0
210.4
84.5
218.7
40.1
242.2
96.8
89.6
87.8
82.1
30.5
51.5
86.7
37.2
26.9
216.6
56.5
42.8
4126
3902
3164
349.2
4444
344.2
3973
2438
2313
210.9
40.6
45.3
502.8
191.7
116.0.
18.9
105.8
19.5
42.6
3785
952.3
211.9
473
17
AAZ
4-Cl
6.8
697.2
250
149
SLT [36,37]
88.4
455
n.t.
8900
>50000
374
7.0
a
Mean from 3 different assays by a stopped-flow technique (errors were in the range of 5e10% of the reported values).
ꢁ Better inhibition data were obtained for the most abundant hCA
0.09 and 0.07 respectively). Adequate SIs (i.e., 0.36e0.46) were also
calculated for the medium nanomolar VchCA- inhibitors such as
the unsubstituted 1, the 4-isopropyl 6, the 3-chlorophenyl 9 and the
bulky tert-butylphenyl 15 (see Table 2). As expected, compounds
II isozyme as the KI values reported in Table 1 ranged between
26.9 and 242.2 nM. The best performing inhibitors among the
sulfanilamide bearing series resulted in the trifluoromethyl
compound 4 (KI 40.1 nM) followed by the methyl 2, methoxy 6,
isopropyl 7, perfluoro 8 or 3-chloro 9 containing moieties (KI
values in the range of 82.1e96.8 nM). The remaining 1, 3, 5
afforded rather poor inhibition potencies (KI values 210.4, 218.7,
242.2 nM). Among the longer ethylbenzenesulfonamide de-
rivatives 10e17, the most active compounds resulted in the
phenyl unsubstituted 10, the trifluoromethyl 13 and the nitro 14
derivatives with KI values of 30.5, 37.2 and 26.9 nM respectively.
Conversely, introduction in 10 of a 4-chloro, 4-methyl, 2,6-
difluoro, 4-fluoro and 4-tert-butyl moiety (as in 17, 11, 16, 12
and 15) decreased the hCA II inhibition potency up to 1.4-, 1.7-,
1.9-, 2.8- and 7.1-fold, respectively.
a
1e17 resulted ineffective to discriminate either the VchCA-
over the off-target hCA II, whereas pentafluorophenyl derivative 7
showed slight selective inhibition for the BpsCA- (SI 0.91). Better
results were obtained when the BpsCA- SI values were considered.
As reported in Table 2, the unsubstituted compound 1 was the best
in exerting its activity on the BpsCA- over the hCA II (SI 0.18)
b and g
b
g
g
although such a feature was mainly ascribed to the ineffectiveness
in inhibiting the latter (KIs of 37 and 210.4 nM, respectively in
Table 1). The most potent BpsCA-g inhibitors, identified from ki-
netic data in Table 1 (i.e. 4, 14 and 16), showed mild SI values (i.e.
0.5, 0.7 and 0.3 respectively) and comparable with medium nano-
molar inhibitors such as the fluoro 4 and the tert-butyl 15
derivatives.
The ability of compounds to specifically interact on bacterial CAs
over the human expressed ones, is the major parameter to be
considered when selection of chemical moieties for future devel-
opment as antimicrobial drugs is operated. Therefore, Selectivity
Indexes (SI; KI bacterial CA/KI hCA II) of our compounds were
calculated and reported in Table 2. The most abundant and kinet-
ically efficient hCA II was considered as a reference off-target iso-
form with the intent to identify chemical species possessing
elevated target discrimination.
3. Conclusion
To the best of our knowledge, this is the first report dealing with
the molecular hybridization approach applied to the well-known
anti-infective Taurolidine 1. We merged the active metabolite
1,2,4-thiadiazinane-1,1-dioxide core (Taurultam 3) within small
molecules bearing the primary arylsulfonamide moiety and thus
intended to act as inhibitors of the bacterial expressed CAs. A set of
17 new compounds was synthesized and profiled in vitro against
the Gram-negative Vibrio cholerae and Burkholderia pseudomallei
Data in Table 2 referred to the VchCA-a allowed to identify that
the most potent inhibitors against such an isoform in vitro (i.e.,
compounds 4, 5, 7, 10, 12 and 16 in Table 1) also showed signifi-
cative SI values and ranging between 0.04 and 0.24. Specifically, the
4-nitro 5, the pentafluorophenyl derivative 7 and the longer 4-
fluoro substituted ethylsulfanilamide 12 were particularly effec-
expressed CAs (i.e. VchCA-
BpsCA- ). The results clearly showed effective inhibition of the
VchCA- (i.e., compounds 4, 5, 7, 10, 12 and 16) and BpsCA- (i.e.,
compounds 4, 14 and 16) isoforms with KIs in the low nanomolar
range. Satisfactory kinetic data were observed for the VchCA- , as
a, VchCA-b, VchCA-g, BpsCA-b and
g
a
g
tive in discriminating between VchCA-a over the hCA II (SIs of 0.04,
b
4

O. Akgul, A. Angeli, S. Selleri et al.
European Journal of Medicinal Chemistry 219 (2021) 113444
Table 2
Selectivity indexes (SI) for titled compounds 1e17.
Selectivity Index (SI)a
Comp
VchCA-
a
/hCA II
VchCA-
b/hCA II
VchCA-
g/hCA II
BpsCA-
b
/hCA II
BpsCA-g/hCA II
0.36
5.95
2.29
0.24
0.04
0.84
0.09
6.75
0.46
0.24
17.4
0.07
21.8
3.49
0.44
0.14
15.9
2.05
4.06
18.9
97.3
13.1
3.6
49.6
3.92
48.4
79.9
44.9
2.43
1.09
1.58
17.5
16.9
4.95
34.7
77.4
29.3
222.4
29.6
94.6
90.5
87.5
82.5
282.6
165.5
94.9
232.9
318.7
41.0
3.1
7.1
0.18
1.4
0.3
0.5
3.9
0.44
9.2
8.0
0.83
1.5
9.8
2.2
3.1
0.7
0.5
0.3
16.3
19.5
15.2
1.80
48.0
0.91
4.7
5.1
2.7
119.9
2.6
6.7
204.5
1.3
7.0
156.0
207.7
1.5
a
SIs are indicative of isozyme selectivity: selective inhibitors show low values.
compounds 13 and 14 resulted medium nanomolar inhibitors. The
appropriate VchCA-a SIs reported, perfectly matched with the most
the reaction was finished (TLC monitoring), the mixture was
filtered and the solvent was evaporated under vacuum. The
resulting crude products were purified by silica gel column chro-
matography eluting with the appropriate solvent system to afford
the titled compounds D1-D17.
potent inhibitors against this class (i.e., compounds 4, 5, 7, 10, 12
and 16) and thus allowing to easily identify promising compounds
for future development. Although this study lacks evidences on the
1,2,4-thiadiazinane-1,1-dioxide to retain its disassemble feature
when incorporated into more complex molecules, it is reasonable
to assume that data here reported successfully sustain our idea of
designing a new generation of CA-based antibiotics also endowed
with old fashioned and free of resistance mechanism of actions.
4.1.2. 4-((2-(Benzylamino)ethyl)sulfonamido)benzenesulfonamide
D1
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt C1,
benzyl chloride (1.0 equiv.), Cs2CO3 (1.0 equiv) and DMF (8.0 ml)
were treated according to the general procedure A. The resulting
crude product was purified by silica gel column chromatography
eluting with EtOAc: MeOH 3:0.1 to afford D1 in 3% yield. m.p:
4. Experimental section
153.7 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d: 3.21e3.25 (m, 2H, eCH2),
4.1. Chemistry
3.64e3.68 (m, 2H, eCH2), 4.11 (s, 2H, benzylic-CH2), 7.37 (s, 2H,
SO2NH2), 7.38e7.51 (m, 7H, AreH), 7.82 (d, J ¼ 8.8 Hz, 2H, AreH),
Anhydrous solvents and all reagents were purchased from
Sigma-Aldrich and Interlab (Germany). All reactions involving air-
or moisture-sensitive compounds were performed in an argon at-
mosphere using dried glassware. 1H and 13C NMR spectra were
scanned on an Agilent 600 MHz Premium COMPACT NMR spec-
trometer using DMSO‑d6 as a solvent. Chemical shifts were re-
ported in parts per million (ppm) and the coupling constants (J)
were expressed in Hertz (Hz). Splitting patterns were designated as
follows: s, singlet; d, doublet; t, triplet; m, multiplet; brs, broad
singlet. Infrared spectra were run on a Perkin Elmer Spectrum 100
FT-IR equipped with a Universal ATR Sampling Accessory and the
frequencies were expressed in cmꢀ1. Analytical thin-layer chro-
matography (TLC) was carried out on Merck silica gel F-254 plates.
Melting points were taken with Stuart® (SMP30) melting point
apparatus in open capillary tubes and were uncorrected. High-
resolution mass spectrometry (HR-MS) analyses were performed
on Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS.
11.2 (s, 1H). 13C NMR (100 MHz, DMSO‑d6)
d: 142.1, 139.5, 139.5,
129.5, 129.2, 128.3, 127.9, 118.9, 59.9, 48.6, 47.1. HRMS (ESI-MS): m/z
[MþH]þ calcd for C15H20N3O4S2: 370.0890; Found: 370.0871.
4.1.3. 4-((2-((4-Methylbenzyl)amino)ethyl)sulfonamido)
benzenesulfonamide D2
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt C1, 4-
methylbenzyl chloride (1.0 equiv.), Cs2CO3 (1.0 equiv) and DMF
(8.0 ml) were treated according to the general procedure A. The
resulting crude product was purified by silica gel column chroma-
tography eluting with EtOAc: MeOH (3:0.1) to afford D2 in 1% yield.
m.p: 175 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d: 2.25 (s, 3H, CH3),
3.21e3.22 (m, 2H, eCH2), 3.37e3.39 (m, 2H, eCH2), 3.78 (s, 2H,
benzylic-CH2,), 7.12 (d, J ¼ 7.8 Hz, 2H, AreH), 7.20 (d, J ¼ 8.8 Hz, 2H,
AreH), 7.36 (s, 2H, SO2NH2), 7.57 (d, J ¼ 8.6 Hz, 2H, AreH), 7.78 (d,
J ¼ 8.6 Hz, 2H, AreH), 11.3 (s, 1H). 13C NMR (100 MHz, DMSO‑d6)
d:
137.0, 135.5, 132.6, 128.8, 128.2, 126.7, 124.1, 119.5, 62.2, 49.4, 40.9,
33.1. HRMS (ESI-MS): m/z [MþH]þ calcd for C16H22N3O4S2,
384.1046; Found: 384.1044.
4.1.1. General procedure A for the synthesis of compounds D1-D17
[22,23]
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt C1
(1.0 equiv.) or 4-(2-((2-aminoethyl)sulfonamido)ethyl)benzene-
sulfonamide HCl salt C2 (1.0 equiv.), the appropriate benzyl chlo-
ride (1.0 equiv.) and Cs2CO3 (1.0 equiv.) were dissolved in dry
dimethylformamide (DMF, 8.0 ml) and stirred at r.t. for 48 h. Once
4.1.4. 4-((2-((4-Fluorobenzyl)amino)ethyl)sulfonamido)
benzenesulfonamide D3
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt C1, 4-
flourobenzyl chloride (1.0 equiv.), Cs2CO3 (1.0 equiv.) and DMF
5

O. Akgul, A. Angeli, S. Selleri et al.
European Journal of Medicinal Chemistry 219 (2021) 113444
(8.0 ml) were treated according to the general procedure A. The
resulting crude product was purified by silica gel column chroma-
tography eluting with EtOAc: MeOH 3:0.1 to afford D3 in 7% yield.
chromatography eluting with EtOAc: MeOH (3:0.1) to afford D7 in
8% yield. m.p: 176.7 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d: 2.86e2.88
(m, 2H, eCH2), 3.35e3.39 (m, 2H, eCH2), 3.81 (m, 2H, benzylic-
CH2,), 7.34e7.34 (m, 4H, AreH, SO2NH2), 7.79 (d, J ¼ 8.6 Hz, 2H,
m.p: 193.7 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d: 2.88e2.91 (m, 2H,
eCH2), 3.35e3.39 (m, 2H, eCH2), 3.67 (m, 2H, benzylic-CH2),
7.12e7.16 (m, 2H, AreH), 7.30e7.36 (m, 6H, AreH, SO2NH2),
7.78e7.80 (d, J ¼ 8.6 Hz, 2H, AreH), 11.2 (s, 1H). 13C NMR (100 MHz,
AreH), 11.1 (s, 1H). 13C NMR (100 MHz, DMSO‑d6; 19F decoupled)
d:
144.8, 142.0, 139.9, 137.2, 131.0, 128.2, 119.0, 112.0, 62.0, 43.4, 40.9.
HRMS (ESI-MS): m/z [MþH]þ calcd for C15H15F5N3O4S2, 460.0419;
Found: 460.04151.
DMSO‑d6)
d
: 163.4 (d, J1 ¼ 245.57 Hz, C40), 141.6, 140.2, 133.4 (d,
CF
J2 ¼ 8.4 Hz, 30), 131.0, 128.2, 119.7, 116.5 (d, J3 ¼ 22.07 Hz, C2’),
CF
CF
60.1, 48.2, 41.4. HRMS (ESI-MS): m/z [MþH]þ calcd for
4.1.9. 4-((2-((4-Methoxybenzyl)amino)ethyl)sulfonamido)
benzenesulfonamide D8
C
15H19FN3O4S2, 388.0723; Found: 388.0730.
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt C1, 4-
methoxybenzyl chloride (1.0 equiv.), Cs2CO3 (1.0 equiv) and DMF
(8.0 ml) were treated according to the general procedure A. The
resulting crude product was purified by silica gel column chroma-
tography eluting with EtOAc: MeOH (3:0.1) to afford D8 in 3% yield.
4.1.5. 4-((2-((4-(Trifluoromethyl)benzyl)amino)ethyl)sulfonamido)
benzenesulfonamide D4
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt C1, 4-
(trifluoromethyl)benzyl chloride (1.0 equiv.), Cs2CO3 (1.0 equiv) and
DMF (8.0 ml) were treated according to the general procedure A.
The resulting crude product was purified by silica gel column
chromatography eluting with EtOAc: MeOH (3:0.1) to afford D4 in
m.p: 133.8 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d: 2.88e2.92 (m, 2H,
eCH2), 3.34e3.38 (m, 2H, eCH2), 3.63 (s, 2H, benzylic-CH2), 3.76 (s,
3H, OCH3), 6.88 (d, J ¼ 8.5 Hz, 2H, AreH), 7.20 (d, J ¼ 8.5 Hz, 2H,
AreH), 7.28 (d, 2H, SO2NH2), 7.32 (d, J ¼ 8.8 Hz, 2H, AreH), 7.79 (t,
1% yield. 1H NMR (400 MHz, DMSO‑d6)
d: 2.89e2.92 (m, 2H, eCH2),
3.11e3.57 (m, 2H, eCH2), 3.78 (s, 2H, benzylic-CH2), 7.30 (s, 2H,
SO2NH2), 7.33e7.35 (m, 2H, AreH), 7.52 (d, J ¼ 7.8 Hz, 2H, AreH),
J ¼ 7.7 Hz, 2H, AreH), 11.2 (s, 1H). 13C NMR (100 MHz, DMSO‑d6)
d:
159.1, 142.9, 139.0, 132.6, 130.2, 128.2, 118.9, 114.5, 55.9, 62.5, 51.8,
43.4. HRMS (ESI-MS): m/z [MþH]þ calcd for C16H22N3O5S2
400.0995; Found: 400.1001.
7.69e7.70 (m, 2H, AreH), 7.79e7.81 (m, 2H, AreH), 11.2 (s, 1H). 13
C
NMR (100 MHz, DMSO‑d6) d
: 141.6, 140.2, 133.4, 131.0, 129.4 (d, J1
CF
¼ 245.58 Hz), 128.2, 127.4, 119.7, 116.5, 60.0, 48.2, 41.4. HRMS (ESI-
MS): m/z [MþH]þ calcd for C16H19F3N3O4S2, 438.0691; Found:
438.0695.
4.1.10. 4-((2-((3-Chlorobenzyl)amino)ethyl)sulfonamido)
benzenesulfonamide D9
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt C1, 3-
chlorobenzyl chloride (1.0 equiv.), Cs2CO3 (1.0 equiv) and DMF
(8.0 ml) were treated according to the general procedure A. The
resulting crude product was purified by silica gel column chroma-
tography eluting with EtOAc: MeOH (3:0.1) and crystallised from
ButOH to afford D9 in 2% yield. m.p: 165.5 ꢂC. 1H NMR (400 MHz,
4.1.6. 4-((2-((4-Nitrobenzyl)amino)ethyl)sulfonamido)
benzenesulfonamide D5
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt C1, 4-
nitrobenzyl chloride (1.0 equiv.), Cs2CO3 (1.0 equiv) and DMF
(8.0 ml) were treated according to the general procedure A. The
resulting crude product was purified by silica gel column chroma-
tography eluting with EtOAc: MeOH (3:0.1) to afford D5 in 2% yield.
DMSO‑d6) d: 3.33e3.39 (m, 2H, eCH2), 3.67e3.71 (m, 2H, eCH2),
4.27 (s, 2H, benzylic-CH2), 7.37e7.40 (m, 4H, AreH, SO2NH2),
7.49e7.52 (m, 3H, AreH), 7.66 (s, 1H, AreH), 7.84 (d, J ¼ 8.7 Hz, 2H,
1H NMR (400 MHz, DMSO‑d6)
d: 2.93e2.97 (m, 2H, eCH2),
3.41e3.45 (m, 2H, eCH2), 3.83 (s, 2H, benzylic-CH2), 7.33e7.35 (m,
4H, AreH, SO2NH2), 7.57 (d, J ¼ 8.4 Hz, 2H, AreH), 7.84 (d, J ¼ 8.6 Hz,
2H, AreH), 8.19 (d, J ¼ 8.8 Hz, 2H, AreH), 11.2 (s, 1H). 13C NMR
AreH), 11.2 (s, 1H). 13C NMR (100 MHz, DMSO‑d6)
d: 140.2, 137.9,
134.6, 131.3, 130.9, 130.0, 127.0, 126.1, 126.0, 116.0, 62.1, 52.0, 38.6.
HRMS (ESI-MS): m/z [MþH]þ calcd for C15H19ClN3O4S2, 404.0500;
Found: 404.05024.
(100 MHz, DMSO‑d6) d: 146.3, 140.2, 133.4, 131.0, 128.2, 127.4, 119.7,
116.5, 59.9, 48.2, 41.4. HRMS (ESI-MS): m/z [MþH]þ calcd for
C
15H19N4O6S2, 415.0668; Found: 415.0670.
4.1.11. 4-(2-((2-(Benzylamino)ethyl)sulfonamido)ethyl)
benzenesulfonamide D10
4.1.7. 4-((2-((4-Isopropylbenzyl)amino)ethyl)sulfonamido)
benzenesulfonamide D6
4-(2-((2-Aminoethyl)sulfonamido)ethyl)benzenesulfonamide
HCl salt C2 (1.0 equiv.), benzyl chloride (1.0 equiv.), Cs2CO3 (1.0
equiv) and DMF (8.0 ml) were treated according to the general
procedure A. The resulting crude product was purified by silica gel
column chromatography eluting with EtOAc: MeOH: ACN
(3:0.2:0.2) to afford D10 in 4% yield as an oil. 1H NMR (400 MHz,
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt C1, 4-
isopropylbenzyl chloride (1.0 equiv.), Cs2CO3 (1.0 equiv) and DMF
(8.0 ml) were treated according to the general procedure A. The
resulting crude product was purified by silica gel column chroma-
tography eluting with EtOAc: MeOH (3:0.1) to afford D6 in 6% yield.
DMSO‑d6) d: 2.84e2.89 (m, 4H, eCH2), 3.18e3.24 (m, 4H, eCH2),
m.p: 172 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d: 0.98 (s, 3H, isopropyl-
3.74 (s, 2H, benzylic-CH2), 7.32 (m, 2H, SO2NH2), 7.34e7.35 (m, 2H,
AreH), 7.36 (s, 2H, AreH), 7.45 (d, J ¼ 8.3 Hz, 3H, AreH), 7.79 (d,
J ¼ 8.4, 2H, AreH), 7.99 (s, 1H, SO2NH-). 13C NMR (100 MHz,
CH3), 1.00 (s, 4H, isopropyl-CH3, -CH-), 2.62e2.68 (m, 2H, eCH2),
3.13e3.17 (m, 2H, eCH2), 3.43 (s, 2H, benzylic-CH2), 6.70e6.96 (m,
4H, AreH), 7.11 (d, J ¼ 8.6 Hz, 2H, AreH), 7.33e7.35 (m, 4H, AreH,
SO2NH2), 7.58 (d, J ¼ 8.6 Hz, 2H, AreH), 7.76 (s, 1H, SO2NH-), 11.2
DMSO‑d6) d: 144.1, 143.2, 141.3, 130.3, 129.2, 128.9, 127.7, 126.7, 59.4,
51.7, 44.3, 43.8, 36.5. HRMS (ESI-MS): m/z [MþH]þ calcd for
(s, 1H). 13C NMR (100 MHz, DMSO‑d6)
d: 140.2, 137.4, 133.4, 131.0,
C17H23N3O4S2 398.1203; Found: 398.12209.
128.2, 127.4, 119.7, 116.5, 59.9, 48.2, 41.4, 33.1, 23.2. HRMS (ESI-MS):
m/z [MþH]þ calcd for C18H26N3O4S2, 412.1286; Found: 412.1289.
4.1.12. 4-(2-((2-((4-Methylbenzyl)amino)ethyl)sulfonamido)ethyl)
benzenesulfon amide D11
4.1.8. 4-((2-(((Perfluorophenyl)methyl)amino)ethyl)sulfonamido)
benzenesulfon amide D7
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt C1,
perfluorobenzyl chloride (1.0 equiv.), Cs2CO3 (1.0 equiv) and DMF
(8.0 ml) were treated according to the general procedure A. The
resulting crude product was purified by silica gel column
4-(2-((2-Aminoethyl)sulfonamido)ethyl)benzenesulfonamide
HCl salt C2 (1.0 equiv.), 4-methylbenzyl chloride (1.0 equiv.), Cs2CO3
(1.0 equiv) and DMF (8.0 ml) were treated according to the general
procedure A. The resulting crude product was purified by silica gel
column chromatography eluting with EtOAc: MeOH: ACN
(3:0.2:0.2) to afford D11 in 2% yield. 1H NMR (400 MHz, DMSO‑d6)
6

O. Akgul, A. Angeli, S. Selleri et al.
European Journal of Medicinal Chemistry 219 (2021) 113444
d: 2.30 (s, 3H, eCH3), 2.82e2.88 (m, 4H, eCH2), 3.16e3.24 (m, 4H,
DMSO‑d6)
d: 1.32 (s, 9H, tert-butyl-CH3), 2.87e2.90 (m, 4H, CH2),
CH2), 3.68 (s, 2H, benzylic-CH2), 7.15 (d, J ¼ 7.9, 2H, AreH), 7.23 (d,
J ¼ 7.9, 2H, AreH), 7.32 (brs, 2H, SO2NH2), 7.44 (d, J ¼ 8.3, 2H, AreH),
7.79 (d, J ¼ 8.3, 2H, AreH), 7.99 (s, 1H, SO2NH-). 13C NMR (100 MHz,
3.17e3.20 (m, 4H, CH2), 3.82 (m, 2H, benzylic-CH2), 7.30 (s, 2H,
SO2NH2), 7.44 (d, J ¼ 8.1 Hz, 2H, AreH), 7.60 (d, J ¼ 8.0 Hz, 2H,
AreH), 7.70 (d, J ¼ 8.1 Hz, 2H, AreH), 7.79 (d, J ¼ 8.0 Hz, 2H, AreH),
DMSO‑d6)
d
: 143.8, 143.2, 136.8, 130.3, 129.2, 128.9, 127.7, 126.7,
7.99 (s, 1H, SO2NH-). 13C NMR (100 MHz, DMSO‑d6)
d: 150.1, 143.1,
58.9, 51.7, 44.3, 43.8, 36.5, 23.1. HRMS (ESI-MS): m/z [MþH]þ calcd
140.1, 145.0, 143.0, 135.2, 129.8, 126.4, 60.1, 52.4, 48.9, 44.2, 40.7,
36.5, 34.4. HRMS (ESI-MS): m/z [MþH]þ calcd for C21H31N3O4S2
453.1756; Found: 453.1755.
for C18H25N3O4S2 412.1286; Found: 412.1288.
4.1.13. 4-(2-((2-((4-Fluorobenzyl)amino)ethyl)sulfonamido)ethyl)
benzenesulfonamide D12
4.1.17. 4-(2-((2-((2,6-Difluorobenzyl)amino)ethyl)sulfonamido)
ethyl)benzenesulfonamide D16
4-(2-((2-Aminoethyl)sulfonamido)ethyl)benzenesulfonamide
HCl salt C2 (1.0 equiv.), 4-fluorobenzyl chloride (1.0 equiv.), Cs2CO3
(1.0 equiv) and DMF (8.0 ml) were treated according to the general
procedure A. The resulting crude product was purified by silica gel
column chromatography eluting with EtOAc: MeOH: ACN
(3:0.2:0.2) to afford D12 in 4% yield. 1H NMR (400 MHz, DMSO‑d6)
4-(2-((2-Aminoethyl)sulfonamido)ethyl)benzenesulfonamide
HCl salt C2 (1.0 equiv.), 2,6-difluorobenzyl chloride (1.0 equiv.),
Cs2CO3 (1.0 equiv) and DMF (8.0 ml) were treated according to the
general procedure A. The resulting crude product was purified by
silica gel column chromatography eluting with EtOAc: MeOH: ACN
(3:0.2:0.2) to afford D16 in 11% yield. m.p: 115 ꢂC. 1H NMR
d: 2.84e2.93 (m, 4H, eCH2), 3.22e3.25 (m, 4H, eCH2), 4.80 (s, 2H,
benzylic-CH2,), 7.16e7.21 (m, 2H, AreH), 7.32 (s, 2H, SO2NH2),
7.40e7.46 (m, 4H, AreH), 7.79 (d, J ¼ 8.2 Hz, 2H, AreH), 7.99 (s, 1H,
(400 MHz, DMSO‑d6) d: 2.87e2.90 (m, 4H, CH2), 3.17e3.20 (m, 4H,
CH2), 3.82 (m, 2H, benzylic-CH2), 7.12e7.19 (m, 2H, AreH), 7.33 (s,
2H, SO2NH2), 7.40 (d, J ¼ 8.2 Hz, 2H, AreH), 7.50e7.56 (m, 2H,
AreH), 7.78 (d, J ¼ 8.2 Hz, 2H, AreH), 7.99 (s, 1H, SO2NH-). 13C NMR
SO2NH-). 13C NMR (100 MHz, DMSO‑d6)
d
: 161.2 (d, J1
¼
CF
245.57 Hz), 143.2, 133.4 (d, J3 ¼ 8.4 Hz), 130.3, 128.9, 127.7, 126.7,
CF
116.5 (d, J2 ¼ 22.07 Hz), 62.0, 51.7, 44.3, 43.8, 36.5. HRMS (ESI-
(100 MHz, DMSO‑d6 decoupled from 19F)
d: 162.0, 143.1, 133.5,
CF
MS): m/z [MþH]þ calcd for C17H22FN3O4S2 415.1036 Found415.1032.
130.2, 126.7, 118.8, 112.3, 70.2, 49.8, 49.7, 49.5, 46.5, 35.4. HRMS
(ESI-MS): m/z [MþH]þ calcd for C17H21F2N3O4S2 434.0942; Found:
434.0938.
4.1.14. 4-(2-((2-((4 (Trifluoromethyl)benzyl)amino)ethyl)
sulfonamido)ethyl)benzenesulfonamide D13
4-(2-((2-Aminoethyl)sulfonamido)ethyl)benzenesulfonamide
HCl salt C2 (1.0 equiv.), 4-(trifluoromethyl)benzyl chloride (1.0
equiv.), Cs2CO3 (1.0 equiv) and DMF (8.0 ml) were treated according
to the general procedure A. The resulting crude product was puri-
fied by silica gel column chromatography eluting with EtOAc:
MeOH: ACN (3:0.2:0.2) to afford D13 in 3% yield. m.p: 148 ꢂC. 1H
4.1.18. 4-(2-((2-((4-Chlorobenzyl)amino)ethyl)sulfonamido)ethyl)
benzenesulfonamide D17
4-(2-((2-Aminoethyl)sulfonamido)ethyl)benzenesulfonamide
HCl salt C2 (1.0 equiv.), 4-chlorobenzyl chloride (1.0 equiv.), Cs2CO3
(1.0 equiv) and DMF (8.0 ml) were treated according to the general
procedure A. The resulting crude product was purified by silica gel
column chromatography eluting with EtOAc: MeOH: ACN
(3:0.2:0.2) to afford D17 in 10% yield. m.p: 124 ꢂC. 1H NMR
NMR (400 MHz, DMSO‑d6) d: 2.84e2.88 (m, 4H, eCH2), 3.19e3.24
(m, 4H, eCH2), 3.83 (m, 2H, benzylic-CH2), 7.32 (s, 2H, SO2NH2),
7.45 (d, J ¼ 8.1 Hz, 2H, AreH), 7.59 (d, J ¼ 8.0 Hz, 2H, AreH), 7.71 (d,
J ¼ 8.1 Hz, 2H, AreH), 7.80 (d, J ¼ 8.0 Hz, 2H, AreH), 7.99 (s, 1H,
(400 MHz, DMSO‑d6) d: 2.87e2.90 (m, 4H, CH2), 3.17e3.20 (m, 4H,
CH2), 3.82 (m, 2H, benzylic-CH2), 7.12e7.19 (m, 2H, AreH), 7.33 (s,
2H, SO2NH2), 7.40 (d, J ¼ 8.2 Hz, 2H, AreH), 7.50e7.56 (m, 2H,
AreH), 7.78 (d, J ¼ 8.2 Hz, 2H, AreH), 7.99 (s, 1H, SO2NH-). 13C NMR
SO2NH-). 13C NMR (100 MHz, DMSO‑d6)
d: 143.9, 143.2, 135.1, 130.2,
130.1, 129.4 (d, J1 ¼ 245.58 Hz), 128.0, 128.3, 124.9, 58.8, 44.3, 40.9,
CF
36.3, 34.9. HRMS (ESI-MS): m/z [MþH]þ calcd for C18H22F3N3O4S2
(100 MHz, DMSO‑d6) d: 142.6,140.9,138.3,132.6,130.3,128.6,128.3,
466.1077; Found: 466.1081.
128.0, 64.3, 52.4, 43.2, 38.1, 34.2. HRMS (ESI-MS): m/z [MþH]þ calcd
for C17H22ClN3O4S2 432.0740; Found: 432.0744.
4.1.15. 4-(2-((2-((4-Nitrobenzyl)amino)ethyl)sulfonamido)ethyl)
benzenesulfonamide D14
4.1.19. General procedure B for the synthesis of compounds 1e17
[24]
4-(2-((2-Aminoethyl)sulfonamido)ethyl)benzenesulfonamide
HCl salt C2 (1.0 equiv.), 4-nitrobenzyl chloride (1.0 equiv.), Cs2CO3
(1.0 equiv) and DMF (8.0 ml) were treated according to the general
procedure A. The resulting crude product was purified by silica gel
column chromatography eluting with EtOAc: MeOH: ACN
(3:0.2:0.2) to afford D14 in 7% yield. m.p: 152 ꢂC. 1H NMR (400 MHz,
D1-D17 (1.0 equiv.) were dissolved in MeOH (2 ml) and the
solution was adjusted to pH ¼ 13 with 5% NaOH aqueous solution
followed by addition of 37% formaldehyde. The reaction mixture
was subjected to ultrasound irradiation for 8 h, was quenched with
water, extracted with EtOAc and the combined organic layers dried
over anhydrous sodium sulfate (Na2SO4), filtered, and concentrated
under vacuum. The crude products were purified by silica gel col-
umn chromatography and additionally preparative thin layer
chromatography to afford 4-(4-(substitutedbenzyl)-1,1-dioxido-
1,2,4-thiadiazinan-2-yl)benzenesulfonamide derivatives (1e9) as
white solids and 4-(2-(4-(substitutedbenzyl)-1,1-dioxido-1,2,4-
thiadiazinan-2-yl)ethyl) benzenesulfonamide derivatives (10e17)
as dark yellow oils or yellow solids.
DMSO‑d6) d: 2.86e2.90 (m, 4H, CH2), 3.17e3.20 (m, 4H, CH2), 3.80
(m, 2H, benzylic-CH2), 7.31 (s, 2H, SO2NH2), 7.44 (d, J ¼ 8.1 Hz, 2H,
AreH), 7.60 (d, J ¼ 8.0 Hz, 2H, AreH), 7.70 (d, J ¼ 8.1 Hz, 2H, AreH),
7.79 (d, J ¼ 8.0 Hz, 2H, AreH), 7.99 (s, 1H, SO2NH-). 13C NMR
(100 MHz, DMSO‑d6) d: 146.3, 145.7, 143.0, 135.2, 131.2, 130.0, 129.8,
126.4, 58.9, 44.2, 40.7, 36.5, 34.4. HRMS (ESI-MS): m/z [MþH]þ calcd
for C17H22N4O6S2 442.0981; Found: 442.09872.
4.1.16. 4-(2-((2-((4-(tert-Butyl)benzyl)amino)ethyl)sulfonamido)
ethyl)benzenesulfonamide D15
4.1.20. 4-(4-Benzyl-1,1-dioxido-1,2,4-thiadiazinan-2-yl)
benzenesulfonamide 1
4-(2-((2-Aminoethyl)sulfonamido)ethyl)benzenesulfonamide
HCl salt C2 (1.0 equiv.), 4-tert-butylbenzyl chloride (1.0 equiv.),
Cs2CO3 (1.0 equiv) and DMF (8.0 ml) were treated according to the
general procedure A. The resulting crude product was purified by
silica gel column chromatography eluting with EtOAc: MeOH: ACN
(3:0.2:0.2) to afford D15 in 10% yield. m.p: 87 ꢂC. 1H NMR (400 MHz,
4-((2-(Benzylamino)ethyl)sulfonamido)benzenesulfonamide
D1 (1.0 equiv.) was treated with MeOH and 37% formaldehyde at
pH ¼ 13 according to the general procedure B. The crude product
was purified by silica gel column chromatography eluting with
EtOAc: Hxn (1:1) to afford the titled compound 1 as a white solid in
7

O. Akgul, A. Angeli, S. Selleri et al.
European Journal of Medicinal Chemistry 219 (2021) 113444
10% yield. mp: 191 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d: 3.28e3.30
benzylic-CH2), 4.61 (s, 2H,.-CH2), 7.60e7.87 (m, 8H, AreH, SO2NH2),
(m, 2H, eCH2), 3.44e3.47 (m, 2H, eCH2), 3.89 (s, 2H, benzylic-CH2),
4.54 (s, 2H, eCH2), 7.38e7.40 (m, 7H, AreH, SO2NH2), 7.63 (d, J ¼
8.6 Hz, 2H, AreH), 7.84 (d, J ¼ 8.5 Hz, 2H, AreH). 13C NMR (100 MHz,
8.24e8.26 (m, 2H, AreH). 13C NMR (100 MHz, DMSO‑d6)
d: 147.8,
146.6, 144.1, 130.9, 129.8, 127.6, 127.4, 124.5, 73.5, 55.1, 50.2, 47.8.
HRMS (ESI-MS): m/z [MþH]þ, calculated for C16H18N4O6S2
426.0668; found 426.0671.
DMSO‑d6) d: 141.8,139.9,130.5,130.0,129.6,128.2,127.4,119.5, 73.0,
60.1, 59.8, 48.2. HRMS (ESI-MS): m/z [MþH]þ, calculated for
C
16H19N3O4S2 382.0890; found 382.0887.
4.1.25. 4-(4-(4-Isopropylbenzyl)-1,1-dioxido-1,2,4-thiadiazinan-2-
yl)benzenesulfon amide 6
4.1.21. 4-(4-(4-Methylbenzyl)-1,1-dioxido-1,2,4-thiadiazinan-2-yl)
benzenesulfonamide 2
4-((2-((4-Isopropylbenzyl)amino)ethyl)sulfonamido)benzene-
sulfonamide D6 (1.0 equiv) was treated with MeOH and 37%
formaldehyde at pH ¼ 13 according to the general procedure B. The
crude product was purified by silica gel column chromatography
eluting with EtOAc: Hxn (1:1) to afford the titled compound 6 as a
white solid in 10% yield. mp: 88.6 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
4-((2-((4-Methylbenzyl)amino)ethyl)sulfonamido)benzene-
sulfonamide D2 (1.0 equiv.) was treated with MeOH and 37%
formaldehyde at pH ¼ 13 according to the general procedure B. The
crude product was purified by silica gel column chromatography
eluting with EtOAc:Hxn (1:1) to afford the titled compound 2 as a
d: 1.19e1.22 (m, 6H, isopropyl-CH3), 2.88 (m, 1H, isopropyl-CH-),
white solid in 5% yield. mp: 183 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d:
3.25e3.26 (m, 2H, eCH2), 3.44e3.45 (m, 2H, eCH2), 3.84 (s, 2H,
benzylic-CH2), 4.55 (s, 2H, CH2), 7.23e7.25 (m, 2H, AreH),
7.28e7.30 (m, 2H, AreH), 7.44 (s, 2H, SO2NH2), 7.58e7.67 (m, 2H,
AreH), 7.76e7.81 (m, 1H, AreH), 7.83e7.85 (m, 1H, AreH). 13C NMR
2.30 (s, 3H, eCH3), 3.27e3.28 (m, 2H, eCH2), 3.43e3.44 (m, 2H,
eCH2), 3.84 (s, 2H, benzylic-CH2), 4.53 (s, 2H, eCH2), 7.16e7.26 (m,
4H, AreH), 7.40 (brs, 2H, SO2NH2), 7.58e7.64 (m, 2H, AreH),
7.76e7.87 (m, 2H, AreH). 13C NMR (100 MHz, DMSO‑d6)
d: 141.8,
(100 MHz, DMSO‑d6) d: 148.5, 144.3, 142.8, 130.1, 129.9, 127.4, 127.3,
139.9, 130.5, 130.0, 129.6, 128.2, 127.4, 119.5, 73.0, 60.1, 59.8, 48.2,
33.1. HRMS (ESI-MS): m/z [MþH]þ, calculated for C17H21N3O4S2
396.0973; found 396.1011.
127.1, 73.1, 55.7, 50.1, 47.8, 34.1, 24.8. HRMS (ESI-MS): m/z [MþH]þ,
calculated for C19H25N3O4S2 423.1286; found 423.1285.
4.1.26. 4-(1,1-Dioxido-4-((perfluorophenyl)methyl)-1,2,4-
thiadiazinan-2-yl)benzenesulfonamide 7
4.1.22. 4-(4-(4-Fluorobenzyl)-1,1-dioxido-1,2,4-thiadiazinan-2-yl)
benzenesulfonamide 3
4-((2-(((Perfluorophenyl)methyl)amino)ethyl)sulfonamido)
benzenesulfonamide D7 (1.0 equiv) was treated with MeOH and
37% formaldehyde at pH ¼ 13 according to the general procedure B.
The crude product was purified by silica gel column chromatog-
raphy eluting with EtOAc: Hxn (1:1) to afford the titled compound 7
as a white solid in 38% yield. mp: 91.8 ꢂC. 1H NMR (400 MHz,
4-((2-((4-Fluorobenzyl)amino)ethyl)sulfonamido)benzene-
sulfonamide D3 (1.0 equiv.) was treated with MeOH and 37%
formaldehyde at pH ¼ 13 according to the general procedure B. The
crude product was purified by silica gel column chromatography
eluting with EtOAc: Hxn (1:1) to afford the titled compound 3 as a
white solid in 3% yield. mp: 68.5 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d:
DMSO‑d6) d: 3.32e3.33 (m, 2H, eCH2), 3.43e3.44 (m, 2H, eCH2),
3.28e3.29 (m, 2H, eCH2), 3.46e3.47 (m, 2H, eCH2), 3.88 (s, 2H,
benzylic-CH2), 4.50e4.59 (m, 2H, eCH2), 7.17e7.21 (m, 2H, AreH),
7.40 (brs, 2H, SO2NH2), 7.41e7.44 (m, 2H, AreH), 7.59e7.65 (m, 2H,
4.05 (s, 2H, benzylic-CH2), 4.59 (s, 2H,.-CH2), 7.43 (s, 2H, SO2NH2),
7.65 (d, J ¼ 8.4 Hz, 2H, AreH), 7.85 (d, J ¼ 8.4 Hz, 2H, AreH). 13C NMR
(100 MHz, DMSO‑d6 decoupled from 19F)
d: 144.8, 142.0, 139.9,
137.2, 131.1,128.2,119.4,116.0, 72.5, 50.3, 47.9, 43.5. HRMS (ESI-MS):
m/z [MþH]þ, calculated for C16H14F5N3O4S2 471.0346; found
471.0341.
AreH), 7.79e7.89 (m, 2H, AreH). 13C NMR (100 MHz, DMSO‑d6)
d:
163.5 (d, J1 ¼ 245.89 Hz), 143.6 (d, J2 ¼ 211.9 Hz), 134.4 (d,
CF
CF
J4 ¼ 2.92 Hz),131.9 (d, J3 ¼ 8.12 Hz),128.4,127.4,116.1,115.8, 73.1,
CF
CF
55.1, 50.0, 47.8. HRMS (ESI-MS): m/z [MþH]þ, calculated for
C
16H18FN3O4S2 400.0796; found 400.0795.
4.1.27. 4-(4-(4-Methoxybenzyl)-1,1-dioxido-1,2,4-thiadiazinan-2-
yl)benzenesulfonamide 8
4.1.23. 4-(1,1-Dioxido-4-(4-(trifluoromethyl)benzyl)-1,2,4-
thiadiazinan-2-yl)benzenesulfonamide 4
4-((2-((4-Methoxybenzyl)amino)ethyl)sulfonamido)benzene-
sulfonamide D8 (1.0 equiv) was treated with MeOH and 37%
formaldehyde at pH ¼ 13 according to the general procedure B. The
crude product was purified by silica gel column chromatography
eluting with EtOAc: Hxn (1:1) to afford the titled compound 8 as a
white solid in 10% yield. mp: 94.6 ꢂC. 1H NMR (400 MHz, DMSO- d6)
4-((2-((4-(Trifluoromethyl)benzyl)amino)ethyl)sulfonamido)
benzenesulfonamide D4 (1.0 equiv) was treated with MeOH and
37% formaldehyde at pH ¼ 13 according to the general procedure B.
The crude product was purified by silica gel column chromatog-
raphy eluting with EtOAc: Hxn (1:1) to afford the titled compound 4
as a white solid in 32% yield. mp: 160.5 ꢂC. 1H NMR (400 MHz,
d: 3.25e3.26 (m, 2H, eCH2), 3.44e3.45 (m, 2H, eCH2), 3.76 (m, 3H,
eOCH3), 3.81 (s, 2H, benzylic-CH2), 4.51e4.52 (m, 2H, CH2),
6.92e6.94 (m, 2H, AreH), 7.27e7.30 (m, 2H, AreH), 7.44 (s, 2H,
SO2NH2), 7.58e7.63 (m, 2H, AreH), 7.77e7.84 (m, 2H, AreH). 13C
DMSO‑d6)
d: 3.30e3.32 (m, 2H, eCH2), 3.50 (brs, 2H, eCH2), 4.01 (s,
2H, benzylic-CH2), 4.57 (s, 2H, eCH2), 7.40 (brs, 2H, SO2NH2),
7.59e7.80 (m, 8H, AreH). 13C NMR (100 MHz, DMSO‑d6)
d: 142.3,
NMR (100 MHz, DMSO- d6) d: 159.6, 142.5, 139.4, 132.1, 130.2, 128.9,
140.1, 133.4, 131.1, 129.3 (d, J1 ¼ 245.58 Hz), 128.2, 127.4, 119.7,
118.9, 114.5, 72.9, 62.5, 51.8, 43.4. HRMS (ESI-MS): m/z [MþH]þ,
CF
116.5, 73.3, 55.2, 50.1, 47.8. HRMS (ESI-MS): m/z [MþH]þ, calculated
calculated for C17H21N3O5S2 411.0923; found 411.0917.
for C17H18F3N3O4S2 449.0691; found 449.0688.
4.1.28. 4-(4-(3-Chlorobenzyl)-1,1-dioxido-1,2,4-thiadiazinan-2-yl)
benzenesulfonamide 9
4.1.24. 4-(4-(4-Nitrobenzyl)-1,1-dioxido-1,2,4-thiadiazinan-2-yl)
benzenesulfonamide 5
4-((2-((3-Chlorobenzyl)amino)ethyl)sulfonamido)benzene-
sulfonamide D9 (1.0 equiv) was treated with MeOH and 37%
formaldehyde at pH ¼ 13 according to the general procedure B. The
crude product was purified by silica gel column chromatography
eluting with EtOAc: Hxn (1:1) to afford the titled compound 9 as a
white solid in 23% yield. mp: 68.1 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
4-((2-((4-Nitrobenzyl)amino)ethyl)sulfonamido)benzenesulfo-
namide D5 (1.0 equiv) was treated with MeOH and 37% formalde-
hyde at pH ¼ 13 according to the general procedure B. The crude
product was purified by silica gel column chromatography eluting
with e EtOAc: Hxn (1:1) to afford the titled compound 5 as a white
solid in 21% yield. mp: 118.3 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d:
d: 3.28 (m, 2H, eCH2), 3.48 (m, 2H, eCH2), 3.92 (s, 2H, benzylic-
3.28e3.29 (m, 2H, eCH2), 3.49e3.50 (m, 2H, eCH2), 4.07 (s, 2H,
CH2), 4.57 (m, 2H,.-CH2), 7.34e7.48 (m, 4H, AreH), 7.44 (s, 2H,
8

O. Akgul, A. Angeli, S. Selleri et al.
European Journal of Medicinal Chemistry 219 (2021) 113444
SO2NH2), 7.59e7.65 (m, 2H, AreH), 7.78e7.87 (m, 2H, AreH). 13C
preparative thin layer chromatography with EtOAc: Hxn (2.5:1) to
NMR (100 MHz, DMSO‑d6)
d
: 144.1, 140.9, 134.1, 133.9, 129.7, 129.3,
afford the titled compound 13 as a dark yellowish solid in 25% yield.
128.6, 128.4, 127.5, 127.4, 73.3, 55.2, 50.1, 47.8. HRMS (ESI-MS): m/z
m.p: 78 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d: 2.88e2.89 (m, 2H, CH2),
[MþH]þ, calculated for C16H18ClN3O4S2 415.0427; found 415.0430.
3.0 (m, 2H, CH2), 3.26 (m, 2H, CH2), 3.48e3.50 (m, 2H, CH2), 3.81 (s,
2H, benzylic-CH2), 4.07e4.10 (m, 2H, eCH2), 7.34e7.41 (m, 2H,
AreH), 7.59e7.61 (m, 2H, AreH), 7.69e7.81 (m, 4H, AreH). 13C NMR
4.1.29. 4-(2-(4-Benzyl-1,1-dioxido-1,2,4-thiadiazinan-2-yl)ethyl)
benzenesulfonamide 10
(100 MHz, DMSO‑d6) d
: 143.9,140.4,133.8, 130.5,130.2,129.7 (d, J1
CF
4-(2-((2-(Benzylamino)ethyl)sulfonamido)ethyl)benzenesulfo-
namide D10 (1 eqv) was treated with MeOH and 37% formaldehyde
at pH ¼ 13 according to the general procedure B. The crude product
was purified by silica gel column chromatography eluting with
EtOAc: Hxn (1:1) followed by preparative thin layer chromatog-
raphy with EtOAc: Hxn (2.5:1) to afford the titled compound 10 as a
¼ 245.58 Hz), 126.7, 126.0, 124.9, 70.1, 56.2, 49.9, 49.5, 46.3, 35.4.
HRMS (ESI-MS): m/z [MþH]þ, calculated for C19H22F3N3O4S2
477.1004; found 477.1007.
4.1.33. 4-(2-(4-(4-Nitrobenzyl)-1,1-dioxido-1,2,4-thiadiazinan-2-
yl)ethyl)benzenesulfonamide 14
dark yellowish oil in 5% yield. 1H NMR (400 MHz, DMSO‑d6)
d: 2.84
(brs, 2H, CH2), 2.99 (brs, 2H, CH2), 3.24 (s, 2H, eCH2), 3.47 (s, 2H,
eCH2), 3.70 (s, 2H, benzylic-CH2), 4.02 (m, 2H, eCH2), 7.36e7.38 (m,
9H, AreH, SO2NH2), 7.69e7.81 (m, 2H, AreH). 13C NMR (100 MHz,
4-(2-((2-((4-Nitrobenzyl)amino)ethyl)sulfonamido)ethyl)ben-
zenesulfonamide D14 (1.0 equiv) was treated with MeOH and 37%
formaldehyde at pH ¼ 13 according to the general procedure B. The
crude product was purified by silica gel column chromatography
eluting with EtOAc: Hxn (1:1) followed by preparative thin layer
chromatography with EtOAc: Hxn (2.5:1) to afford the titled com-
pound 14 as a dark yellowish solid in 19% yield. m.p: 104.2 ꢂC. 1H
DMSO‑d6) d: 143.9, 143.1, 138.6, 130.2, 129.8, 129.7, 129.4, 128.4,
126.7, 78.0, 69.9, 57.1, 50.0, 49.5, 46.5. HRMS (ESI-MS): m/z [MþH]þ,
calculated for C18H23N3O4S2 409.1130; found 409.1127.
4.1.30. 4-(2-(4-(4-Methylbenzyl)-1,1-dioxido-1,2,4-thiadiazinan-
2yl)ethyl)benzenesulfonamide 11
NMR (400 MHz, DMSO‑d6) d: 2.84e2.86 (m, 2H, CH2), 2.96 (m, 2H,
CH2), 3.22 (m, 2H, CH2), 3.45 (m, 2H, CH2), 3.82 (s, 2H, benzylic-
CH2), 4.04e4.06 (m, 2H, CH2), 7.28e7.36 (m, 2H, AreH), 7.34 (s, 2H,
SO2NH2), 7.60e7.73 (m, 4H, AreH), 8.17e8.21 (m, 2H, AreH). 13C
4-(2-((2-((4-Methylbenzyl)amino)ethyl)sulfonamido)ethyl)
benzenesulfonamide D11 (1.0 equiv) was treated with MeOH and
37% formaldehyde at pH ¼ 13 according to the general procedure B.
The crude product was purified by silica gel column chromatog-
raphy eluting with EtOAc: hexane (1:1) followed by preparative
thin layer chromatography with EtOAc: Hxn (2.5:1) to afford the
titled compound 11 as a yellowish solid in 15% yield. m.p: 174.1 ꢂC.
NMR (100 MHz, DMSO‑d6) d: 147.8, 147.2, 143.9, 143.1, 130.7, 130.3,
126.7, 124.5, 70.4, 56.0, 49.9, 49.5, 46.4, 35.5. HRMS (ESI-MS): m/z
[MþH]þ, calculated for C18H22N4O6S2 457.0981; found 454.0978.
1H NMR (400 MHz, DMSO- d6)
d: 2.34 (s, 3H, CH3), 2.83e2.86 (m,
4.1.34. 4-(2-(4-(4-(tert-Butyl)benzyl)-1,1-dioxido-1,2,4-
2H, CH2), 2.98 (m, 2H, CH2), 3.22e3.25 (m, 2H, CH2), 3.45e3.49 (m,
2H, CH2), 3.67 (s, 2H, benzylic-CH2), 4.03 (m, 2H, eCH2), 7.20 (d,
J ¼ 7.5 Hz, 2H, AreH), 7.26 (d, J ¼ 7.0 Hz, 2H, AreH), 7.34 (s, 2H,
thiadiazinan-2-yl)ethyl)benzene sulfonamide 15
4-(2-((2-((4-(tert-Butyl)benzyl)amino)ethyl)sulfonamido)ethyl)
benzenesulfonamide D15 (1.0 equiv) was treated with MeOH and
37% formaldehyde at pH ¼ 13 according to the general procedure B.
The crude product was purified by silica gel column chromatog-
raphy eluting with EtOAc: Hxn (1:1) followed by preparative thin
layer chromatography with EtOAc: Hxn (2.5:1) to afford the titled
compound 15 as a dark yellowish oil in 5% yield. 1H NMR (400 MHz,
SO2NH2), 7.37 (d, J ¼ 7.0 Hz, 2H, AreH), 7.77e7.79 (m, 2H, AreH). 13
C
NMR (100 MHz, DMSO- d6) d: 143.8, 143.1, 137.4, 135.5, 130.2, 129.9,
129.7, 126.7, 69.9, 56.8, 49.9, 49.4, 46.5, 35.4, 21.7. HRMS (ESI-MS):
m/z [MþH]þ, calculated for C19H25N3O4S2 423.1286; found
423.1284.
DMSO‑d6) d: 1.32 (s, 9H, CH3), 2.81e2.85 (m, 2H, CH2), 2.99e3.0 (m,
4.1.31. 4-(2-(4-(4-Fluorobenzyl)-1,1-dioxido-1,2,4-thiadiazinan-2-
yl)ethyl)benzenesulfonamide 12
2H, CH2), 3.23e3.26 (m, 2H, CH2), 3.45e3.49 (m, 2H, CH2), 3.67 (s,
2H, benzylic-CH2), 4.03 (m, 2H, CH2), 7.29e7.43 (m, 8H, AreH;
4-(2-((2-((4-Fluorobenzyl)amino)ethyl)sulfonamido)ethyl)ben-
zenesulfonamide D12 (1.0 equiv) was treated with MeOH and 37%
formaldehyde at pH ¼ 13 according to the general procedure B. The
crude product was purified by silica gel column chromatography
eluting with EtOAc: hexane (1:1) followed by preparative thin layer
chromatography with EtOAc: Hxn (2.5:1) to afford the titled com-
pound 12 as a dark yellowish solid in 6% yield. m.p: 97.2 ꢂC. 1H NMR
SO2NH2), 7.76e7.78 (m, 2H, AreH). 13C NMR (100 MHz, DMSO‑d6)
d:
150.7, 143.9, 143.2, 135.6, 130.2, 129.6, 126.7, 126.2, 69.9, 56.8, 50.0,
49.4, 46.5, 35.4, 35.2, 32.2. HRMS (ESI-MS): m/z [MþH]þ, calculated
for C22H31N3O4S2 465.1756; found 465.1755.
4.1.35. 4-(2-(4-(2,6-Difluorobenzyl)-1,1-dioxido-1,2,4-
(400 MHz, DMSO- d6)
d: 2.86e2.88 (m, 2H, CH2), 2.99e3.0 (m, 2H,
thiadiazinan2yl)ethyl)benzenesulfonamide 16
CH2), 3.25 (m, 2H, CH2), 3.47 (m, 2H, CH2), 3.69 (s, 2H, benzylic-
CH2), 4.03e4.04 (m, 2H, eCH2), 7.19e7.24 (m, 2H, AreH), 7.34 (s, 2H,
SO2NH2), 7.38e7.40 (m, 4H, AreH), 7.70e7.78 (m, 2H, AreH). 13C
4-(2-((2-((2,6-Difluorobenzyl)amino)ethyl)sulfonamido)ethyl)
benzenesulfonamide D16 (1.0 equiv) was treated with MeOH and
37% formaldehyde at pH ¼ 13 according to the general procedure B.
The crude product was purified by silica gel column chromatog-
raphy eluting with EtOAc: Hxn (1:1) followed by preparative thin
layer chromatography with EtOAc: Hxn (2.5:1) to afford the titled
compound 16 as a dark yellowish solid in 5% yield. m.p: 140 ꢂC. 1H
NMR (100 MHz, DMSO‑d6)
d
: 162.5 (d, J1 ¼ 237.5 Hz), 143.9, 143.1,
CF
134.9 (d, J3 ¼ 2.86 Hz, CeF), 131.7, 130.2, 126.7, 116.8 (d,
CF
J2 ¼ 22.07 Hz), 70.1, 56.1, 49.8, 49.5, 46.4, 35.5. HRMS (ESI-MS): m/
CF
z [MþH]þ, calculated for C18H22FN3O4S2 427.1036; found 427.1038.
NMR (400 MHz, DMSO‑d6) d: 2.87e2.91 (m, 2H, CH2), 2.98e3.01 (m,
4.1.32. 4-(2-(1,1-Dioxido-4-(4-(trifluoromethyl)benzyl)-1,2,4-
thiadiazinan-2yl)ethyl)benzenesulfonamide 13
2H, CH2), 3.23e3.26 (m, 2H, CH2), 3.47e3.48 (m, 2H, CH2), 3.76 (s,
2H, benzylic-CH2), 4.08 (s, 2H, CH2), 7.12e7.19 (m, 1H, AreH), 7.33
(s, 2H, SO2NH2), 7.40 (d, J ¼ 8.2 Hz, 2H, AreH), 7.50e7.56 (m, 2H,
AreH), 7.78 (d, J ¼ 8.2 Hz, 2H, AreH). 13C NMR (100 MHz, DMSO‑d6
4-(2-((2-((4-(Trifluoromethyl)benzyl)amino)ethyl)sulfona-
mido)ethyl)benzenesulfonamide D13 (1.0 equiv) was treated with
MeOH and 37% formaldehyde at pH ¼ 13 according to the general
procedure B. The crude product was purified by silica gel column
chromatography eluting with EtOAc: Hxn (1:1) followed by
decoupled from 19F)
d: 143.9, 143.1, 133.5, 130.2, 126.7, 121.5, 112.3,
104.9, 70.2, 49.8, 49.7, 49.5, 46.5, 35.4. HRMS (ESI-MS): m/z
[MþH]þ, calculated for C18H21F2N3O4S2 445.0942; found 445.0940.
9

O. Akgul, A. Angeli, S. Selleri et al.
European Journal of Medicinal Chemistry 219 (2021) 113444
4.1.36. 4-(2-(4-(4-Chlorobenzyl)-1,1-dioxido-1,2,4-thiadiazinan-2-
yl)ethyl)benzenesulfonamide 17
Abbreviations used
4-(2-((2-((4-Chlorobenzyl)amino)ethyl)sulfonamido)ethyl)ben-
zenesulfonamide D17 (1.0 equiv) was treated with MeOH and 37%
formaldehyde at pH ¼ 13 according to the general procedure B. The
crude product was purified by silica gel column chromatography
eluting with EtOAc: Hxn (1:1) followed by preparative thin layer
chromatography with EtOAc: Hxn (2.5:1) to afford the titled com-
pound 17 as a dark yellowish solid in 1% yield. m.p: 75.5 ꢂC. 1H NMR
CA
carbonic anhydrase
carbonic anhydrase inhibitor(s)
acetazolamide
Sulthiame.
CAI(s)
AAZ
SLT
Appendix A. Supplementary data
(400 MHz, DMSO‑d6) d: 2.82e2.89 (m, 4H, CH2), 3.41e3.42 (m, 4H,
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113444.
CH2), 4.28 (s, 2H, benzylic-CH2), 4.47 (s, 2H, eCH2), 7.08e7.10 (m,
2H, AreH), 7.23e7.25 (m, 2H, AreH), 7.33e7.35 (m, 2H, AreH), 7.43
(s, 2H, SO2NH2), 7.56e7.77 (m, 2H, AreH). 13C NMR (100 MHz,
References
DMSO- d6) d: 143.9, 138.9, 137.8, 132.9, 131.6, 130.2, 129.4, 126.7,
70.1, 57.1, 56.1, 49.9, 49.5, 46.5. HRMS (ESI-MS): m/z [MþH]þ,
[1] https://www.who.int/health-topics/antimicrobial-resistance (last access 04/
12/2020).
calculated for C18H22ClN3O4S2 443.0740; found 443.0735.
[2] https://www.who.int/health-topics/antimicrobial-resistance (last access 04/
12/2020).
[3] C.T. Supuran, C. Capasso, Antibacterial carbonic anhydrase inhibitors: an up-
date on the recent literature, Expert Opin. Ther. Pat. (2020) 1e20.
[4] A. Aspatwar, M. Hammaren, S. Koskinen, B. Luukinen, H. Barker, F. Carta,
4.2. CA inhibition
ꢁ
An Applied Photophysics stopped-flow instrument was used for
assaying the CA catalyzed CO2 hydration activity [25]. Phenol red
(at a concentration of 0.2 mM) was used as an indicator,
C.T. Supuran, M. Parikka, S. Parkkila, b-CA-specific inhibitor dithiocarbamate
Fc14-584B: a novel antimycobacterial agent with potential to treat drug-
resistant tuberculosis, J. Enzym. Inhib. Med. Chem. 32 (2017) 832e840.
[5] A. Aspatwar, J.Y. Winum, F. Carta, C.T. Supuran, M. Hammaren, M. Parikka,
S. Parkkila, Carbonic anhydrase inhibitors as novel drugs against mycobacte-
working at the absorbance maximum of 557 nm, with 20 mM
rial b-carbonic anhydrases: an update on in vitro and in vivo studies, Mole-
cules 23 (2018) 2911e2925.
[6] A. Aspatwar, V. Kairys, S. Rala, M. Parikka, M. Bozdag, F. Carta, C.T. Supuran,
Hepes (pH 7.4) for a-class, 20 mM TRIS (pH 8.3) for eb and eg
classes as a buffer, and 20 mM Na2SO4 (for maintaining constant
ionic strength), following the initial rates of the CA catalyzed CO2
hydration reaction for a period of 10e100 s. The CO2 concentrations
ranged from 1.7 to 17 mM for the determination of the kinetic
parameters and inhibition constants [26,27]. Enzymes concentra-
tions ranged between 5 and 12 nM. For each inhibitor, at least six
traces of the
initial 5e10% of the reaction were 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 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 prior
to the assay, to allow for the formation of the EeI complex. The
inhibition 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 [28e30].
S. Parkkila, Mycobacterium tuberculosis b-Carbonic Anhydrases: novel targets
for developing antituberculosis drugs, Int. J. Mol. Sci. 20 (2019) 5153.
[7] C.T. Supuran, C. Capasso, Biomedical applications of prokaryotic carbonic
anhydrases, Expert Opin. Ther. Pat. 10 (2018) 745e754.
[8] C.T. Supuran, C. Capasso, An overview of the bacterial carbonic anhydrases,
Metabolites 4 (2017) 56.
[9] F. Caruso, J.W. Darnowski, C. Opazo, A. Goldberg, N. Kishore, E.S. Agoston,
M. Rossi, Taurolidine antiadhesive properties on interaction with E. coli; its
transformation in biological environment and interaction with bacteria cell
wall, PloS One 5 (2010), e8927.
[10] E. Bueno, V. Pinedo, F. Cava, Adaptation of vibrio cholerae to hypoxic envi-
ronments, Front. Microbiol. 11 (2020) 1e12.
[11] A.A. Weil, J.B. Harris, Vibrio cholerae, in: Mol. Med. Microbiol., Elsevier, 2015,
pp. 1079e1098.
[12] C.T. French, P.L. Bulterys, C.L. Woodward, A.O. Tatters, K.R. Ng, J.F. Miller,
Virulence from the rhizosphere: ecology and evolution of Burkholderia
pseudomallei-complex species, Curr. Opin. Microbiol. 54 (2020) 18e32.
[13] B. Currie, Melioidosis: evolving concepts in epidemiology, pathogenesis, and
treatment, Semin. Respir. Crit. Care Med. 36 (2015) 111e125.
[14] https://www.cdc.gov/melioidosis/index.html (last access 04/12/2020).
€
[15] O. Akgül, A. Angeli, D. Vullo, F. Carta, C.T. Supuran, Unconventional amino
acids in medicinal chemistry: first report on taurine merged within carbonic
anhydrase inhibitors, Bioorg. Chem. 103 (2020) 104236.
[16] O.Akgul, S. Singh, J. T. Andring, R. McKenna, S. Selleri, F. Carta, A. Angeli, C.T.
Supuran. Handling drug-target selectivity: a study on ureido containing car-
bonic anhydrase inhibitors. Eur. J. Med. Mhem. In press https://doi.org/10.
1016/j.ejmech.2020.113035.
[17] O. Akgul, F.S. Kilic, K. Erol, V. Pabuccuoglu, Synthesis and anticonvulsant ac-
tivity of some N-phenyl-2- phtalimidoethanesulfonamide derivatives, Arch.
Pharm. (Weinheim) 340 (2007) 656e660.
Funding sources
Ozlem Akgul (O.A.) is grateful to The Scientific and Technical
[18] R. Winterbottom, J.W. Clapp, W.H. Miller, J.P. English, R.O. Roblin, Studies in
chemotherapy. XV. Amides of pantoyltaurine 1, J. Am. Chem. Soc. 69 (1947)
1393e1401.
_
Research Council of Turkey [TUBITAK 117S516] and Ege University
[16-ECZ-012] which partially funded this work.
€
_
€
[19] O. Akgül, I. Oztürk, A. Aygül, S¸ . Ermertcan, Synthesis and antimicrobial activity
of some taurinamide derivatives, Marmara Pharm. J. 212 (2017) 361e370.
[20] O.M.S. Curley, J.E. McCormick, R.S. McElhinney, T.B.H. McMurry, Intermediates
in the ing-manske reaction, Arkivoc 2003 (2003) 180e189.
Fabrizio Carta (F$C.) is grateful to “Bando di Ateneo per il
Finanziamento di Progetti Competitivi per Ricercatori a Tempo
Determinato (RTD) dell’Universita di Firenze” 2020e202100, and
Fondazione Cassa di Risparmio di Firenze (Grant Number
ECR2018.1001) for partially supporting this work. This work was
also financed by the Italian Ministry of University and Research,
project FISR2019_04819 BacCAD (to CTS and CC).
[21] R.A. Smits, M. Adami, E.P. Istyastono, O.P. Zuiderveld, C.M.E. van Dam, F.J.J. de
Kanter, A. Jongejan, G. Coruzzi, R. Leurs, I.J.P. de Esch, Synthesis and QSAR of
quinazoline sulfonamides as highly potent human histamine H4 receptor
inverse agonists, J. Med. Chem. 53 (2010) 2390e2400.
[22] R.N. Salvatore, C.H. Yoon, K.W. Jung, Synthesis of secondary amines, Tetra-
hedron 57 (2001) 7785e7811.
[23] R.N. Salvatore, A.S. Nagle, W.J. Kyung, Cesium effect: high chemoselectivity in
direct N-alkylation of amines, J. Org. Chem. 67 (2002) 674e683.
[24] R.W. Pfirrmann, Perhydro-1,2,4-thiadiazinedioxides-(1,1) and Their Prepara-
tion, US, vol. 3, 1969, p. 408, 423.
Declaration of competing interest
[25] R.G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase,
J. Biol. Chem. 246 (1971) 2561e2573.
[26] A. Angeli, M. Ferraroni, M. Pinteala, S.S. Maier, B.C. Simionescu, F. Carta, S. Del
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.
Prete, C. Capasso, C.T. Supuran, Crystal structure of a tetrameric type II
b-
10

O. Akgul, A. Angeli, S. Selleri et al.
European Journal of Medicinal Chemistry 219 (2021) 113444
[29] F. Pacchiano, F. Carta, D. Vullo, A. Scozzafava, C.T. Supuran, Inhibition of b-
carbonic anhydrases with ureido-substituted benzenesulfonamides, Bioorg.
Med. Chem. Lett 21 (2011) 102e105.
[30] F. Carta, D. Vullo, A. Maresca, A. Scozzafava, C.T. Supuran, Mono-/dihydrox-
ybenzoic acid esters and phenol pyridinium derivatives as inhibitors of the
mammalian carbonic anhydrase isoforms I, II, VII, IX, XII and XIV, Bioorg. Med.
Chem. 21 (2013) 1564e1569.
carbonic anhydrase from the pathogenic bacterium Burkholderia pseudo-
mallei, Molecules 25 (2020) 2269.
[27] S. Del Prete, S. Isik, D. Vullo, V. De Luca, V. Carginale, A. Scozzafava,
C.T. Supuran, C. Capasso, DNA cloning, characterization, and inhibition studies
of an alpha-carbonic anhydrase from the pathogenic bacterium Vibrio chol-
erae, J. Med. Chem. 23 (2012) 10742e10748.
[28] A. Karioti, F. Carta, C.T. Supuran, Phenols and polyphenols as carbonic anhy-
drase inhibitors, Molecules 21 (2016) 1649.
11