Unconventional amino acids in medicinal chemistry: First report on taurine merged within carbonic anhydrase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104236

This study reports the design, synthesis of a series of taurine containing benzenesulfonamide derivatives which were all screened in vitro against the physiological relevant human (h) expressed Carbonic Anhydrase (CA; EC 4.2.1.1) I, II, IX, XII isozymes. Compound 2, 5, 11–16 displayed superior inhibitory activities against the tumor associated hCA IX over the reference drug Acetazolamide (AAZ). Both hCA IX and XII isoforms were selectively inhibited only by compound 3, whereas the chloro-containing compound 12 was showed as the most selective and effective inhibitor profile for the CA IX isoforms. To the best of our knowledge the data reported herein are the first of this kind and introduce in the literature new compounds worth for future development within the Medicinal Chemistry field.

Full text of DOI:10.1016/j.bioorg.2020.104236

Bioorganic Chemistry 103 (2020) 104236
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Unconventional amino acids in medicinal chemistry: First report on taurine
merged within carbonic anhydrase inhibitors
Özlem Akgül , Andrea Angeli , Daniela Vullo , Fabrizio Cartab,⁎, Claudiu T. Supuranb
b
c
a,⁎
b
c
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ege University, 35100 Bornova, İzmir, Turkey
Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via Della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy
Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
This study reports the design, synthesis of a series of taurine containing benzenesulfonamide derivatives which
were all screened in vitro against the physiological relevant human (h) expressed Carbonic Anhydrase (CA; EC
Taurine
Benzenesulfonamide
4
.2.1.1) I, II, IX, XII isozymes. Compound 2, 5, 11–16 displayed superior inhibitory activities against the tumor
Carbonic Anhydrase Inhibitors (CAIs)
Structure-Activity Relationship (SAR)
associated hCA IX over the reference drug Acetazolamide (AAZ). Both hCA IX and XII isoforms were selectively
inhibited only by compound 3, whereas the chloro-containing compound 12 was showed as the most selective
and effective inhibitor profile for the CA IX isoforms. To the best of our knowledge the data reported herein are
the first of this kind and introduce in the literature new compounds worth for future development within the
Medicinal Chemistry field.
1
. Introduction
Amino acids possess significant roles in medicinal chemistry as free
permeability, acidity, and biological activities [6–8] (Fig. 1).
In mammals taurine is mainly assumed from exogenous sources (i.e.
diet) and only a minor amount is endogenously produced by means of
various pathways in the liver [9,10]. However the ability biosynthesis
of taurine is progressively reduced with aging and thus its assumption
through diet becomes gradually essential. Taurine deficiency was re-
ported in several pathologies such as growth related anomalies, neu-
ronal damage to the retina, kidney dysfunction, diabetes, Alzheimer
and cardiovascular diseases [6,8]. Additional physiological roles of the
taurine account for osmoregulation, neuromodulation, neuroprotection
and radical scavenger among others [6,7]. Unfortunately taurine itself
cannot be used as a drug since its limited permeability, unclear phar-
macokinetics and short halflife [6,11]. Despite a limited number of
studies on taurine derivatives have been reported, molecules endowed
with effective anticancer, antibacterial and antiepileptic properties
were described. Overall there is a persistent lack of structure–activity
relationship (SAR) on taurine containing compounds acting on specific
biological systems [6,12,13].
drugs or being part of more complex structures such as natural sub-
stances, their derivatives or of new synthesis [1–7]. The main ad-
vantage in dealing with amino acids is to aquire readily available
building blocks which contain: i) two orthogonal functional groups
modifiable by convenient and well established synthetic methods (i.e.
acylation, alkylation and amidation); ii) additional moieties further
usable to derivatization and iii) a chiral center, usually a carbon atom,
with major optical differences between eukaryotic or prokaryotic or-
ganisms [1,2]. All such features are packed into low weight molecular
entities. In the vast panorama of the so called “proteinogenic” amino
acids are listed the 22 primary amino acids both coded and noncoded,
the ones obtained from post-translational modifications, or via post-
translational cross-linking of two amino acid entities [1–3]. The non-
proteinogenic amino acids are not included in protein’s primary se-
quences and usually are produced as secondary metabolites in bacteria,
fungi, plants or marine organisms. Finally, many non-naturally occur-
ring amino acids have been synthesized with the aim to introduce new
chemical features in such useful building blocks [1–5].
In light of these considerations, we aimed to investigate the role of
such an amino acid as spacer within linear small molecules designed to
modulate the human expressed metalloenzymes Carbonic Anhydrases
(CAs, EC 4.2.1.1) as schematic represented in Fig. 2.
We turned our attention on the Taurine (β-aminoethansulfonic acid)
which is the only free amino acid present in mammals. It structurally
differs from conventional amino acids as it contains the sulfonic acid
instead of the carboxylic moiety, and this greatly affects taurine’s
According to our strategy the enzymatic ion coordinating moiety
(i.e. the primary aryl sulfonamide) is connected to the taurine with the
secondary sulfonamidic group, whereas its amine is used to install
⁎
Corresponding authors.
E-mail addresses: ozlem.akgul@ege.edu.tr (Ö. Akgül), fabrizio.carta@unifi.it (F. Carta).
https://doi.org/10.1016/j.bioorg.2020.104236
Received 8 June 2020; Received in revised form 5 August 2020; Accepted 10 August 2020
Available online 26 August 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.

Ö. Akgül, et al.
Bioorganic Chemistry 103 (2020) 104236
2
10.4–2362 nM and 77.20–1667 nM respectively. Among the amide
derivatives the tolyl 5 was the most potent (K 210.4 nM) and
I
comparable to the standard drug, AAZ (K 250 nM). The introduc-
I
tion of both the electron withdrawing nitro group (compound 6) and
the bulky electron donating dimethylamino group (compound 8)
resulted with a 3-fold decrease of affinity against the hCA I when
Fig. 1. Chemical structure of Taurine.
compared to the nonsubstituted phenyl 1 (K 798.7 nM). Derivatives
I
variously substituted aryl tail sections by means of an amidic or
thioureidic moiety. Some of us investigated the role of (thio)ureido
containing linkers installed in close proximity of the sulfonamide
warhead [14] (Fig. 3). X-ray crystallographic studies on appropriate
ligands-CA adducts and in vitro kinetic assays proved that the flexibility
of the (thio)ureido moiety was far superior to the amide in allowing the
ligands to adopt energetically more favorable conformations within the
hCA IX enzymatic cleft and thus explaining their preferential inhibition
for such an isoform [14–17]. We thought to make use of the taurine
amino acid to install such moieties (i.e. thiourea/amide) closer to the
tail of the molecule. Tailored end-sections of low molecular weight
compounds endowed with inhibitory features against the CAs, are
fundamental for driving their interaction towards the specific isoforms
of interest [18,19]. Such an approach proved succesfull since it makes
use of interactions occurring between the terminal part of the inhibitors
with the aminoacidic residues composing the rim of the enzymatic cleft
which share very low homology among the various isoforms. The sec-
ondary sulfonamide moiety may be considered as additional point of
interaction of the ligand within the enzyme by means of hydrogen
bonds and then further contributing to the stabilization of the adduct.
2
, 3, 4, 7, 9 and 10 bearing electron-withdrawing (i.e. fluoro, tri-
fluoromethyl), electron donating (i.e. methoxy) and bulky alkyl
groups exhibited similar inhibitory profile with K values ranging
between 620 and 957.5 nM. The introduction of the thiourea moiety
as in 11 enhanced the inhibition potency against the hCA I (K s of
5.0 and 798.7 nM for 11 and 1 respectively). Among this series the
direct incorporation into the phenyl ring of halogens, such as chloro
I
I
9
1
2 and fluoro 13, resulted in 4-, 17-fold decrease of the inhibition
potency when compared to the parent 11. On the other hand the
trifluoro moiety as in 17 greatly enhanced the potency, as such
derivative resulted the most potent among the thiourea bearing
series with a K of 77.2 nM. As for the remaining compound 14–16
I
an almost flat kinetic profile was obtained.
As for the dominant and most active isozyme hCA II better inhibi-
•
tion data were obtained being the K s values of the amide and
I
thioureido series comprised between 5.1 and 715.1 nM and
.0–86.7 nM respectively. The introduction of strong electron
7
withdrawing moieties within the phenyl ring as for compounds 3, 6
and 7 resulted detrimental for the inhibition potency. As reported in
Table 1 the above mentioned derivatives resulted 4.4-, 8.6- and 6.5-
fold less potent inhibitors of the hCA II when compared to the parent
unsubstituted compound 1. Interestingly the inhibition potency was
restored when small moieties endowed with electron donating fea-
tures, either by means of pure electronic or mesomeric effects, to-
ward the phenyl ring were installed as in 2 and 5. As reported in
2
. Results and discussion
2.1. Chemistry
Final compounds 1–17 were obtained by a 5-step linear reaction
Table 1 the chloro containing derivative 2 showed a K
I
value of
approach as reported in Scheme 1.
1
8.1 nM and the tolyl 5 was the most potent inhibitor within this the
The key intermediate 2-(N-(4-sulfamoylphenyl)sulfamoyl)ethana-
mine as hydrochloric salt D was obtained according to synthetic
methods previously described in the literature [20–24]. Taurine was
protected with phtalic anhydride in acetic acid solution under reflux to
afford the 2-phthalimidoethanesulfonic acid A as potassium salt which
was converted to the chlorinated derivative B and subjected to treat-
ment with sulfanilamide in pyridine at 60 °C to afford the intermediate
C. Employment of Ing-Manske procedure to with hydrazine hydrate
solution in ethanol afforded the desired intermediate D in 60% yield.
A series of amide couplings on D with appropriate commercially
available aryl acids by using 1-1′carbonyldiimidazole (CDI), diisopro-
pylethylamine (DIPEA) acetonitrile and water afforded the final com-
pounds 1–10 in 12–48% yields (Scheme 1) [25]. The thiourea deriva-
tives 11–17 were obtained in 18–39% yield from reaction of D with the
various aryl isothiocyanates commercially available or ready to use in
laboratory in the presence of DIPEA and dry acetonitrile (Scheme 1)
amide series with a K
I
of 5.1 nM thus 2.5-fold more potent of the
reference AAZ (K 12.8 nM). The bulkiness of the inserted moieties
I
resulted detrimental for the inhibition potencies as reported in
Table 1 for compounds 8–10. A rather different kinetic profile was
obtained for the thioureido series (Table 1). The introduction of the
thioureidic moiety itself as in 11 determined a 4.3-fold enhance-
ment of the inhibition potency in comparison to the amide con-
taining derivative 1. Both the fluoro and the trifluoro moieties as in
1
3 and 17 respectively were detrimental for the inhibition potency
(
K
I
s of 32.4 and 86.7 nM respectively). Interestingly an enhance-
ment of the K values was also observed for compounds 12, 14 and
I
1
5 which contain electron donating groups. Quite unexpectedly the
nitro derivative (compound 16 in Table 1) was the only moiety
which induced a sensible increase of the inhibition potency against
the hCA II being 2.5-fold more potent when compared to the un-
substituted thioureido progenitor 11 (K s of 7.0 and 17.8 nM re-
I
[
26]. All final compounds were purified by silica gel column chroma-
spectively).
tography using appropriate eluting mixtures followed by trituration or
recrystallization and then were fully characterized by 1H NMR, 13C
NMR, HRMS, FTIR (ATR) spectroscopy and elemental analyses.
Interesting inhibition profile was obtained for the transmembrane
and tumor related hCA IX as reported in Table 1. In analogy to the
hCA I previously discussed the introduction of strong electron
withdrawing groups within the amide series, as for compounds 3, 6
•
2.2. Carbonic anhydrase inhibition
and 7, resulted detrimental for the inhibition potency being the K s
I
associated 1.6, 8.22 and 7.95-fold higher when compared to the
The final compounds 1–10 (amide derivatives) and 11–17 (thiourea
unsubstituted phenyl compound 1. Similarly, the electron donating
derivatives) were profiled in vitro for their inhibitory activities against
the most relevant hCA I, II, IX and XII isozymes by using the stopped-
flow method applied to the carbon dioxide hydration assay [27]. All the
data obtained are reported in Table 1 and refereed to the clinically used
drug acetazolamide (AAZ) as standard.
containing moieties 4, 8 and 9 also showed increased K values of
I
1
38.0, 527.6 and 97.5 nM respectively. Unexpectedly the bulky tert-
butyl group as in 10 restored the inhibition potency, being 1.22-fold
more potent when compared to the unsubstituted phenyl compound
1
I
(K s of 35.0 and 42.8 nM respectively). Quite interesting data were
obtained when the chloro and the methyl groups (i.e. in compounds
and 5) were introduced. Both derivatives resulted low nanomolar
inhibitors of the hCA IX isoform with K s of 6.3 and 4.9 nM
The slow cytosolic isozyme hCA I was inhibited with amides 1–10
•
2
and thiourea 11–17 derivatives with
K s in the range of
I
I
2

Ö. Akgül, et al.
Bioorganic Chemistry 103 (2020) 104236
Fig. 2. Schematic representation of the design strategy adopted.
Fig. 3. General structure of the amide and thiourea derivatives of benzoyltaurinamide derivatives.
respectively, thus significatively more potent than the standard AAZ
as in compounds 16, restored the inhibition potency. As reported in
Table 1 the derivative 16 was the most potent inhibitor of the hCA
(
K of 25.8 nM). As for the thioureido series 11–17 the kinetic profile
I
on the hCA IX accounted for the unsubstituted phenyl derivative 11
and for its chloro containing moiety 12 being potent inhibitors with
IX isoform with a K of 4.9 nM.
I
The kinetic profiling of the entire chemical series synthesized 1–17
•
close K
I
values (K
I
s of 7.2 and 8.0 nM respectively). Compounds 14
on the hCA XII showed a rather homogeneous distribution of the K
I
and 15 which presented the electron donating moieties showed
values in the medium nanomolar range and spanning between 20
close inhibition values (K s of 24.6 and 25.3 nM respectively). In-
I
and 90.6 nM. The only exception was represented by the tri-
terestingly the insertion in 11 of electron withdrawing moieties such
as the fluorine (i.e. 13) and the trifluoromethyl (i.e. 17) spoiled the
inhibition potencies up to 33.9-fold. Unexpectedly the nitro group,
fluoromethyl derivative 17 (K 308.5 nM). Although it is difficult to
I
draw an exhaustive SAR analysis at this stage for such an enzymatic
isoform, a preliminary consideration is on the tert-butyl derivative
Scheme 1. Synthesis of amides 1–10 and thioureas 11–17 derivatives.
3

Ö. Akgül, et al.
Bioorganic Chemistry 103 (2020) 104236
Table 1
Merck (Germany). All other reagents and reactants, including 4-sub-
Stopped-flow kinetic data of compounds 1–17 on hCAs I, II, IX, and XII in
comparison with AAZ. Selectivity indexes (SI) for the inhibition of hCA IX and
XII over hCA I and II are also reported.
stituted phenyl isothiocyanates, were purchased from Sigma-Aldrich
(
(
Germany). Anhydrous solvents were purchased from Sigma-Aldrich
Germany) and Carlo Erba (France). All reactions involving air- or
K
I
(nM)*
moisture-sensitive compounds were performed in a nitrogen atmo-
sphere using dried glassware and syringe techniques to transfer solu-
Comp.
R
CAI
CAII
CAIX
CAXII
Selectivity Index** (SI)
CAIX/CAII
1
tions. H NMR spectra were scanned on a Varian AS 400 Mercury Plus
NMR spectrometer using DMSO‑d as a solvent. Chemical shifts were
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
.
eH
798.7
814.1
957.5
643.5
210.4
77.2
18.1
42.8
6.3
84.2
68.1
90.6
0.6
0.3
reported 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 ex-
.
eCl
.
eFl
337.0 67.0
0.2
.
eOCH
3
28.0
5.1
138.0 36.1
4.9
.
eCH
3
2
4.9
44.4
0.96
0.5
.
eNO
eCF3
eN(CH
2236.0 667.1 345.1 48.0
−1
.
881.1
501.2 332.9 71.2
0.7
pressed in cm . Analytical thin-layer chromatography (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. The solvents used in MS measurements were
ethanol, acetonitrile, and water (LC-MS grade) purchased from Sigma-
Aldrich (Germany). The ESI-MS (Electrospray Ionization-Mass) spectra
were obtained using a Thermo Scientific DSQ II (Austin, TX USA)
equipped with an electrospray source (ESI) operating in both positive
and negative ions. Raw data was collected and processed by Xcalibur™
2.0 software. Elemental analyses (C, H, N, and S) were performed using
a Leco TruSpec CHNS Micro analyzer (Leco Corporation, St. Joseph, MI,
USA) and results were within ± 0.4% of calculated values.
.
3
)
2
2362.0 715.1 527.6 70.8
0.74
0.61
0.99
0.4
.
eCH(CH
3
)
2
765.6
620.7
95.0
159.0 97.5
64.1
20.0
86.0
82.6
65.4
64.1
34.6
30.8
0.
1.
2.
3.
4.
5.
6.
7.
eC(CH
3
)
3
35.3
17.8
48.0
35.0
7.2
eH
eCl
389.4
8.0
0.2
eFl
1667.0 32.4
18.9
24.6
25.3
4.9
0.6
eOCH
3
92.0
97.3
92.2
77.2
250
28.0
32.7
7.0
0.9
eCH
eNO
eCF
3
2
3
0.8
0.7
86.7
12.8
244.1 308.5
2.8
AAZ
25.8
5.7
*
Mean from 3 different assays by a stopped-flow technique (errors were in
the range of ± 5–10% of the reported values).
* The SI ratios (K CAIX/KI CAII) are indicative of isozyme selectivity: a
strong selective inhibitor is characterized by a low value.
*
I
4.2. General procedure A: synthesis of compounds 1–10
The starting compound 2-(N-(4-sulfamoylphenyl)sulfamoyl)etha-
1
0, which is the most potent inhibitor, is worth doing since it is the
namine HCl salt D was obtained by following 4-step reaction previously
described in the literature [19–23]. The various benzoic acids (1.0
equiv.), 1-1′carbonyldiimidazole (1.0 equiv.), and diisopropylethyla-
mine (DIPEA, 2.0 equiv.) were dissolved in dry acetonitrile (ACN,
only compound among the series which allowed to selectively in-
hibit the hCA XII isoform by means of a substitution moiety (i.e. the
tert-butyl) which failed in discriminating among the other isoforms.
8
.0 mL) and stirred at r.t. until the formation of intermediate. 2-(N-(4-
In consideration of the physiological meaning of the isoforms herein
considered we sought to better focus on the hCA IX which is exclusively
sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D was dissolved in
1
0 mL water and added to the reaction mixture. After consumption of
expressed in hypoxic tumors [28]. The selectivity index values (SI; K
I
the starting material, the solvent evaporated. The crude product was
subjected to column chromatography eluting with ethyl acetate/hexane
or ethyl acetate/methanol or dichloromethane/hexane/methanol and
the obtained solid was crystallized or triturated with an appropriate
solvent mixture to afford the final compounds 1–10 [25].
CAIX/K CAII) reported in Table 1 clearly showed the potentiality of our
I
compound series to be further developed as valid theragnostic tools for
the management of such diseases. Among the amide containing series
synthesized 1–10, the aryl halogen derivatives 2 and 3 showed the best
SIs (0.3 and 0.2 for compound 2 and 3 respectively) although they
differ of almost 11-fold their inhibition potencies (K s of 6.3 and
I
4
.3. General procedure B: Synthesis of compounds 11–17
6
7.0 nM for compound 2 and 3 respectively). As for the thioureido
derivatives 11–17 only the chlorophenyl 12 showed good selectivity in
inhibiting the hCA IX (SI 0.2) and a strong potency (K 8.0 nM).
The starting compound 2-(N-(4-sulfamoylphenyl)sulfamoyl)etha-
I
namine HCl salt D was obtained by following a 4-step reaction pre-
viously described in the literature [19–23] 2-(N-(4-sulfamoylphenyl)
sulfamoyl)ethanamine HCl salt D, the various isothiocyanates (1.0
equiv.), and diisopropylethylamine (DIPEA, 2.0 equiv.) were suspended
in dry acetonitrile (ACN, 8.0 mL) and heated at 60 °C under a nitrogen
atmosphere. Once the starting material was consumed, the solvent was
evaporated and the crude product was purified to column chromato-
graphy eluting with ethyl acetate/hexane and the obtained solid was
crystallized or triturated with an appropriate solvent mixture to afford
the final compounds 11–17 [26].
3
. Conclusion
To the best of our knowledge this is the first report on the design,
synthesis and in vitro kinetic profiling of series of taur-
a
inamidobenzensulfonamide derivatives as inhibitors of the physiologi-
cally relevant hCA isoforms I, II, IX, and XII. Our preliminary in-
vestigation clearly showed that the shift of the amide or thioureido
moiety towards the end tail of our molecules allowed to obtain potent
and selective inhibitors against the tumor associated hCA IX. Among
the compounds obtained the derivatives 2, 3 and 12 were the best in
both potency and isoform selectivity and thus pave the way for further
development of our researches in this direction.
4
.3.1. N-(2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethyl)benzamide 1
2
-(N-(4-sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, water
(
10 mL), benzoic acid (1.0 equiv.), 1-1′carbonyldiimidazole (1.0
equiv.), diisopropylethylamine (DIPEA, 2.0 equiv.), dry acetonitrile
(ACN, 8.0 mL) were treated according to the general procedure A. The
crude product was purified by silica gel column chromatography
eluting with dichloromethane/hexane/methanol (5/3/1) and crystal-
4
. Experimental section
4
.1. Chemistry
lized from ethanol to afford the title compound 1 as a white solid in
1
Phtalic anhydride, potassium acetate, anilin, 4-methyl benzoic acid,
-chlorobenzoic acid, and 4-nitrobenzoic acid were purchased from
21% yield. mp: 195.5 °C; H NMR (400 MHz, DMSO‑d
6
) δ: 3.40–3.42
4
(m, 2H, CH
2
), 3.58–3.60 (m, 2H, CH
2
), 7.22 (s, 2H, SO
2
NH ), 7.28 (d,
2
4

Ö. Akgül, et al.
Bioorganic Chemistry 103 (2020) 104236
1
3
J = 8.7 Hz, 2H, Ar-H), 7.39 (t, J = 7.5 Hz, 2H, Ar-H), 7.46–7.48 (m,
C NMR (100 MHz, DMSO‑d
6
) δ: 166.8 (C]O), 162.6, 142.1, 139.8,
1
H, CONH), 7.72–7.70 (m, 5H, Ar-H), 8.53 (t, J = 5.3 Hz, 1H, SO
2
NH).
129.9 (2 X Ar-C), 128.2 (2 X Ar-C), 127.1, 119.3 (2 X Ar-C), 114.5 (2 X
1
3
C NMR (100 MHz, DMSO‑d
6
) δ: 167.3 (C]O), 142.1, 139.7, 134.8,
Ar-C), 56.3, 51.2 (eNHCH
2
CH
2
SO
2
−), 35.1 (eNHCH
2
CH
2
SO −). IR
2
−1
1
32.3, 129.2 (3 X Ar-C), 128.2 (2 X Ar-C), 128.0 (2 X Ar-C), 119.3, 51.0
υmax (cm ): 3371, 3319, 3265 (NeH stretching), 1633 (amide I band),
−
1
(
eNHCH
2
CH
2
SO
2
−), 35.1 (eNHCH
2
CH
2
SO
2
−). IR υmax (cm ): 3342,
1504 (amide II band), 1316 (SO
2
asymmetric stretching), 1138 (SO
2
−
3
309, 3110 (NeH stretching), 1615 (amide I band), 1532 (amide II
symmetric stretching). m/z (ESI negative): 412.27 [M–H] . Elemental
band), 1327 (SO
2
asymmetric stretching), 1145 (SO
2
symmetric
analysis calculated (%) for 0.1 C
3
H
8
O X C16
H
19
N
3
O
6
S : C, 46.67; H,
2
−
stretching). m/z (ESI negative): 382.25 [M–H] . Elemental analysis
4.76; N, 10.02; S, 15.29; found: C, 46.28; H, 4.74; N, 9.69; S, 14.90.
calculated (%) for 0.4 C
2
H
6
O X C15
H
17
N
3
O
5
S : C, 47.22; H, 4.87; N,
2
1
0.46; S, 15.96; found: C, 47.41; H, 4.63; N, 10.49; S, 15.60.
4.3.5. 4-Methyl-N-(2-(N-(4-sulfamoylphenyl)sulfamoyl)ethyl)benzamide
5
4
.3.2. 4-Chloro-N-(2-(N-(4-sulfamoylphenyl)sulfamoyl)ethyl)benzamide 2
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, water
(10 mL), 4-methylbenzoic acid (1.0 equiv.), 1-1′carbonyldiimidazole
(1.0 equiv.), diisopropylethylamine (DIPEA, 2.0 equiv.), dry acetoni-
trile (ACN, 8.0 mL) were treated according to the general procedure A.
The crude product was purified by silica gel column chromatography
eluting with dichloromethane/methanol (5/0.1) and crystallized from
2
-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, water
(
(
10 mL), 4-chlorobenzoic acid (1.0 equiv.), 1-1′carbonyldiimidazole
1.0 equiv.), diisopropylethylamine (DIPEA, 2.0 equiv.), dry acetoni-
trile (ACN, 8.0 mL) were treated according to the general procedure A.
The crude product was purified by silica gel column chromatography
eluting with dichloromethane/hexane/methanol (5/3/1) to afford the
ethanol to afford the title compound 5 as a white solid in 17% yield.
1
1
title compound 2 as a white solid in 33% yield. mp: 233.7 °C; H NMR
mp: 224.1 °C; H NMR (400 MHz, DMSO‑d
6
) δ: 2.29 (m, 3H, CH ),
3
(
400 MHz, DMSO‑d
6
) δ: 3.42–3.44 (m, 2H, CH
2
), 3.59–3.62 (m, 2H,
3.38–3.40 (m, 2H, CH
2
), 3.56–3.59 (m, 2H, CH ), 7.20 (d, J = 8.0 Hz,
2
CH
2
), 7.25 (s, 2H, SO
2
NH
2
), 7.30 (t, J = 8.7 Hz, 2H, Ar-H), 7.50 (m,
2H, Ar-H), 7.22 (s, 2H, SO
2
NH ), 7.29 (d, J = 8.6 Hz, 2H, Ar-H), 7.62
2
2
H, Ar-H), 7.73 (t, J = 9.0 Hz, 4H, Ar-H), 8.64 (t, J = 5.3 Hz, 1H,
(d, J = 8.0 Hz, 2H, Ar-H), 7.71 (d, J = 8.5 Hz, 2H, Ar-H), 8.47 (t,
1
3
13
SO
2
NH). C NMR (100 MHz, DMSO‑d
6
) δ: 166.3 (C]O), 142.1, 139.7,
J = 5.3 Hz, 1H, CONH), 10.35 (brs, 1H, SO
2
NH). C NMR (100 MHz,
1
37.2, 133.6, 129.9 (2 X Ar-C), 129.3 (2 X Ar-C), 128.2, 119.3 (2 X Ar-
DMSO‑d ) δ: 167.2 (C]O), 142.2, 142.2, 139.7, 132.1, 129.8 (2 X Ar-
6
C), 50.9 (eNHCH
2
CH
2
SO
2
−), 35.2 (eNHCH
2
CH
2
SO
2
−). IR υmax
C), 128.2 (2 X Ar-C), 128.1 (2 X Ar-C), 119.3 (2 X Ar-C), 51.1
−
1
−1
(
cm ): 3369, 3315, 3262 (NeH stretching), 1641 (amide I band),
(eNHCH
2
CH
SO
2 2
−), 35.1 (eNHCH
2
CH
2
SO −), 21.9. IR υmax (cm ):
2
1
540 (amide II band), 1334 (SO
2
asymmetric stretching), 1138 (SO
2
3317, 3296, 3108 (NeH stretching), 1611 (amide I band), 1538 (amide
−
symmetric stretching). m/z (ESI negative): 416.13 [M–H] . Elemental
II band), 1327 (SO
2
asymmetric stretching), 1145 (SO
2
symmetric
−
analysis calculated (%) for C15
H
16ClN
3
O
5
S
2
: C, 43.11; H, 3.86; N,
stretching). m/z (ESI negative): 396.24 [M–H] . Elemental analysis
1
0.06; S, 15.34; found: C, 43.47; H, 3.96; N, 10.16; S, 15.71.
calculated (%) for C16
H
19
N
3
O
5
S : C, 48.35; H, 4.82; N, 10.57; S, 16.13;
2
found: C, 48.56; H, 4.98; N, 10.32; S, 16.18.
4
.3.3. 4-Fluoro-N-(2-(N-(4-sulfamoilfenil)sulfamoyl)ethyl)benzamide 3
2
-(N-(4-sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, water
4.3.6. 4-Nitro-N-(2-(N-(4-sulfamoylphenyl)sulfamoyl)ethyl)benzamide 6
2-(N-(4-sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, water
(10 mL), 4-nitrobenzoic acid (1.0 equiv.), 1-1′carbonyldiimidazole (1.0
equiv.), diisopropylethylamine (DIPEA, 2.0 equiv.), dry acetonitrile
(ACN, 8.0 mL) were treated according to the general procedure A. The
crude product was purified by silica gel column chromatography
eluting with ethyl acetate/hexane (3/1) and triturated from di-
(
(
10 mL), 4-fluorobenzoic acid (1.0 equiv.), 1-1′carbonyldiimidazole
1.0 equiv.), diisopropylethylamine (DIPEA, 2.0 equiv.), dry acetoni-
trile (ACN, 8.0 mL) were treated according to the general procedure A.
The crude product was purified by silica gel column chromatography
eluting with ethyl acetate/methanol (5/0.1) to afford the title com-
1
pound 3 as a white solid in 41% yield. mp: 210 °C; H NMR (400 MHz,
DMSO‑d
6
) δ: 3.48–3.51 (m, 2H, CH
2
), 3.65–3.70 (m, 2H, CH
2
),
chloromethane/petroleum ether to afford the title compound 6 as a
1
7
.30–7.34 (m, 4H, Ar-H, SO
2
NH
2
), 7.37 (d, J = 8.7 Hz, 2H, Ar-H), 7.79
white solid in 48% yield. mp: 226.7 °C; H NMR (400 MHz, DMSO‑d
6
)
(
d, J = 8.7 Hz, 2H, Ar-H), 7.85–7.88 (m, 2H, Ar-H), 8.62–8.65 (m, 1H,
δ: 3.51–3.54 (m, 2H, CH
SO NH ), 7.35 (d, J = 8.8 Hz, 2H, Ar-H), 7.78 (d, J = 8.8 Hz, 2H, Ar-
H), 8.01 (d, J = 8.9 Hz, 2H, Ar-H), 8.34 (d, J = 8.9 Hz, 2H, Ar-H),
2
), 3.68–3.73 (m, 2H, CH
2
), 7.31 (s, 2H,
1
3
CONH), 10.41 (brs, 1H, SO
2
NH). C NMR (100 MHz, DMSO‑d
6
) δ:
2
2
1
66.3 (C]O), 163.4 (d, J = 246.70 Hz, C4′), 142.1, 139.8, 131.3 (d,
1
3
J = 3.4 Hz, C2′), 130.7, 130.7, 128.2 (2 X Ar-C), 119.2 (2 X Ar-C),
16.2 (d, J = 21.7 Hz, C3′, C5′), 51.0 (eNHCH CH SO −), 35.2
max (cm ): 3340, 3309, 3114 (NeH
8.94–8.97 (m, 1H, CONH), 10.43 (brs, 1H, SO
(100 MHz, DMSO‑d ) δ: 165.6 (C]O), 150.0, 142.0, 140.4, 139.7,
129.5 (2 X Ar-C), 128.2 (2 X Ar-C), 124.5 (2 X Ar-C), 119.2 (2 X Ar-C),
2
NH).
C NMR
1
2
2
2
6
−
1
(
eNHCH
2
CH
2
2
SO −). IR υ
−
1
stretching), 1623 (amide I band), 1553 (amide II band), 1334 (SO
2
50.8 (eNHCH
2
CH
2
SO
2
−), 35.4 (eNHCH
2
CH
2
SO −). IR υmax (cm ):
2
asymmetric stretching), 1147 (SO
2
symmetric stretching). m/z (ESI
3370, 3320, 3235 (NeH stretching), 1655 (amide I band), 1527 (amide
−
negative): 400.15 [M–H] . Elemental analysis calculated (%) for 0.2
II band), 1305 (SO
2
asymmetric stretching), 1141 (SO
2
symmetric
−
C
H
4 8
O
2
X C15
H
16FN
3
O
5
S
2
: C, 45.29; H, 4.23; N, 10.03; S, 15.30; found:
stretching). m/z (ESI negative): 427.26 [M–H] . Elemental analysis
C, 45.02; H, 4.47; N, 10.36; S, 15.01.
calculated (%) for 0.25 CH
2
Cl
2
X C15
H
16
N
4
O
7
2
S : C, 40.73; H, 3.70; N,
1
2.46; S, 14.26; found: C, 40.66; H, 4.04; N, 12.18; S, 14.25.
4
.3.4. 4-Methoxy-N-(2-(N-(4-sulfamoylphenyl)sulfamoyl)ethyl)benzamide
4
4.3.7. N-(2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethyl)-4-(trifluoromethyl)
benzamide 7
2
-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, water
(
(
10 mL), 4-methoxylbenzoic acid (1.0 equiv.), 1-1′carbonyldiimidazole
1.0 equiv.), diisopropylethylamine (DIPEA, 2.0 equiv.), dry acetoni-
2-(N-(4-sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, water
(10 mL), 4-trifluoromethylbenzoic acid (1.0 equiv.), 1-1′carbonyldii-
midazole (1.0 equiv.), diisopropylethylamine (DIPEA, 2.0 equiv.), dry
acetonitrile (ACN, 8.0 mL) were treated according to the general pro-
cedure A. The crude product was purified by silica gel column chro-
matography eluting with ethyl acetate/methanol (5/0.1) and crystal-
trile (ACN, 8.0 mL) were treated according to the general procedure A.
The crude product was purified by silica gel column chromatography
eluting with ethyl acetate/methanol (5/0.1) and crystallized from iso-
propanol to afford the title compound 4 as a white solid in 39% yield.
1
mp: 221.4 °C; H NMR (400 MHz, DMSO‑d
6
3
) δ: 3.46–3.50 (m, 2H, CH
2
),
lized from isopropanol/water to afford the title compound 7 as a white
1
3
.67–3.69 (m, 2H, CH
Ar-H), 7.30 (s, 2H, SO
t, 4H, Ar-H), 8.46 (t, J = 5.5 Hz, 1H, CONH), 10.40 (brs, 1H, SO
2
2
), 3.84 (s, 3H, OCH ), 7.02 (d, J = 8.8 Hz, 2H,
NH ), 7.37 (d, J = 8.8 Hz, 2H, Ar-H), 7.76–7.81
NH).
solid in 21% yield. mp: 248.3 °C; H NMR (400 MHz, DMSO‑d
6
) δ:
NH ),
7.36–7.38 (m, 2H, Ar-H), 7.79–7.81 (m, 2H, Ar-H), 7.88 (d, J = 8.2 Hz,
2
3.50–3.54 (m, 2H, CH
2
), 3.68–3.73 (m, 2H, CH
2
), 7.31 (s, 2H, SO
2
2
(
2
5

Ö. Akgül, et al.
Bioorganic Chemistry 103 (2020) 104236
2
1
H, Ar-H), 7.99 (d, J = 7.9 Hz, 2H, Ar-H), 8.84–8.87 (m, 1H, CONH),
DMSO‑d
6
) δ: 1.32 (s, 9H, CH
2
2
), 3.47–3.50 (m, 2H, CH ), 3.65–3.70 (m,
2
1
3
0.41 (brs, 1H, SO
2
NH). C NMR (100 MHz, DMSO‑d
6
) δ: 166.2 (C]
2H, CH
2
), 7.30 (s, 2H, SO NH ), 7.37 (d, J = 8.6 Hz, 2H, Ar-H), 7.50 (d,
2
O), 142.1, 139.8, 138.6, 132.2 (q, J = 32 Hz, C-3′), 128.9 (3 X Ar-C),
J = 8.3 Hz, 2H, Ar-H), 7.74 (d, J = 8.3 Hz, 2H, Ar-H), 7.80 (d,
1
4
28.2 (2 X Ar-C), 127.5 (q, J = 272 Hz, C-F3), 126.3 (q, J = 3.6 Hz, C-
J = 8.6 Hz, 2H, Ar-H), 8.53 (t, J = 5.5 Hz, 1H,CONH), 10.41 (brs, 1H,
1
3
′), 119.3 (2
CH SO
X
Ar-C), 50.9 (eNHCH
2
CH
2
SO
2
−), 35.3
SO
2
NH). C NMR (100 MHz, DMSO‑d ) δ: 167.2 (C]O), 155.1, 142.1,
6
−
1
(
eNHCH
2
2
2
−). IR
υ
max (cm ): 3374, 3315, 3262 (NeH
139.7, 132.1, 128.2 (2 X Ar-C), 127.9 (2 X Ar-C), 125.9 (2 X Ar-C),
119.3 (2 X Ar-C), 51.1 (eNHCH CH SO −), 35.5 (eNHCH CH SO −),
35.1, 31.8 (3 X CH ). IR υmax (cm ): 3308, 3205, 3105 (NeH
stretching), 1649 (amide I band), 1545 (amide II band), 1319 (SO
2
2
2
2
2
2
2
−
1
asymmetric stretching), 1138 (SO
2
symmetric stretching). m/z (ESI
3
−
negative): 450.35 [M–H] . Elemental analysis calculated (%) for 0.25
stretching), 1618 (amide I band), 1539 (amide II band), 1329 (SO
2
CH
2
Cl
2
, 0.2 C
4
H
10O X C16
H
16
F
3
N
3
O
5
S
2
: C, 42.01; H, 3.82; N, 8.62; S,
asymmetric stretching), 1146 (SO
2
symmetric stretching). m/z (ESI
−
1
3.15; found: C, 41.93; H, 3.62; N, 8.89; S, 12.84.
negative): 438.39 [M–H] . Elemental analysis calculated (%) for
C
19
H
25
N
3
O
5
S : C, 51.92; H, 5.73; N, 9.56; S, 14.59; found: C, 51.88; H,
2
4
.3.8. 4-Dimethylamino-N-(2-(N-(4-sulfamoylphenyl)sulfamoyl)ethyl)
5.50; N, 9.25; S, 14.61.
benzamide 8
2
-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, water
4.3.11. 4-(2-(3-Phenylthioureido)ethylsulfonamido)benzenesulfonamide
11
(
10 mL), 4-dimethylaminobenzoic acid (1.0 equiv.), 1-1′carbonyldii-
midazole (1.0 equiv.), diisopropylethylamine (DIPEA, 2.0 equiv.), dry
acetonitrile (ACN, 8.0 mL) were treated according to the general pro-
cedure A. The crude product was crystallized from dimethylformamide/
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, phenyl
isothiocyanate (1.0 equiv.), and diisopropylethylamine (DIPEA, 2.0
equiv.), dry acetonitrile (ACN, 8.0 mL) were treated according to the
general procedure B. The crude product was purified by silica gel
column chromatography eluting with ethyl acetate/hexane (3/1) and
ethyl acetate: hexane (2:1) and crystallized from acetonitrile/water to
water to afford the title compound 8 as a white solid in 12% yield. mp:
1
2
3
2
74.2 °C; H NMR (400 MHz, DMSO‑d
6
) δ: 2.99 (s, 6H, 2 X CH ),
3
.44–3.48 (m, 2H, CH
2
), 3.62–3.67 (m, 2H, CH
2
), 6.72 (d, J = 8.8 Hz,
H, Ar-H), 7.30 (s, 2H, SO
2
NH ), 7.38 (d, J = 8.9 Hz, 2H, Ar-H), 7.67
2
afford the title compound 11 as a white solid in 26% yield. mp: 191 °C;
1
(
(
d, J = 8.7 Hz, 2H, Ar-H), 7.80 (d, J = 8.6 Hz, 2H, Ar-H), 8.26–8.23
H NMR (400 MHz, DMSO‑d
6
) δ: 3.54–3.58 (m, 2H, CH
2
), 3.91–3.96
NH ),
7.36–7.38 (m, 6H, Ar-H), 7.74–7.78 (m, 1H, NHCSNH), 7.81 (d,
J = 8.7 Hz, 2H, Ar-H), 9.79 (s, 1H, NHCSNH), 10.41 (brs, 1H, SO NH).
) δ: 181.3 (C]S), 142.29, 139.6, 139.5,
1
3
m, 1H, CONH), 10.36 (brs, 1H, SO
2
NH).
C NMR (100 MHz,
(m, 2H, CH
2
), 7.15–7.19 (m, 1H, Ar-H), 7.30 (s, 2H, SO
2
2
DMSO‑d
6
) δ: 194.9 (C]O), 167.2, 153.1, 142.1, 139.7, 129.4 (2 X Ar-
C), 128.2 (2 X Ar-C), 119.3 (2 X Ar-C), 111.7 (2 X Ar-C), 51.4
2
1
3
(
(
(
eNHCH
2
CH
2
SO
2
−), 34.9 (eNHCH
2
CH
2
2
SO −), 40.6, 39.8. IR υmax
C NMR (100 MHz, DMSO‑d
6
−
1
cm ): 3306, 3102 (NeH stretching), 1604 (amide I band), 1521
129.7 (2 X Ar-C), 128.1 (2 X Ar-C), 125.5, 124.4 (2 X Ar-C), 119.4 (2 X
amide II band), 1328 (SO
2
asymmetric stretching), 1147 (SO
2
sym-
Ar-C), 50.9 (eNHCH
2
CH
2
SO
2
−), 39.6 (eNHCH
2
CH
2
SO −). IR υmax
2
−
−1
metric stretching). m/z (ESI negative): 425.30 [M–H] . Elemental
(cm ): 3374, 3267 (NeH stretching), 1551 (NeH bending), 1318 (SO
2
analysis calculated (%) for 0.2 C
3
H
7
NO X C17
H
22
N
4
O
5
S
2
: C, 47.92; H,
asymmetric stretching), 1241 (C]S stretching), 1142 (SO
2
symmetric
−
5
.35; N, 13.34; S, 14.54; found: C, 47.56; H, 5.33; N, 13.21; S, 14.27.
stretching). m/z (ESI negative): 413.25 [M–H] . Elemental analysis
calculated (%) for 0.1 H
2
O X C15
H
18
N
4
O
4
3
S : C, 43.28; H, 4.41; N, 13.46;
4
.3.9. 4-Isopropyl-N-(2-(N-(4-sulfamoylphenyl)sulfamoyl)ethyl)
S, 23.10; found: C, 43.01; H, 4.52; N, 13.21; S, 22.79.
benzamide 9
2
-(N-(4-sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, water
4.3.12. 4-(2-(3-(4-Chlorophenyl)thioureido)ethylsulfonamido)
benzenesulfonamide 12
(
(
10 mL), 4-isopropylbenzoic acid (1.0 equiv.), 1-1′carbonyldiimidazole
1.0 equiv.), diisopropylethylamine (DIPEA, 2.0 equiv.), dry acetoni-
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, 4-
chlorophenyl isothiocyanate (1.0 equiv.), and diisopropylethylamine
(DIPEA, 2.0 equiv.), dry acetonitrile (ACN, 8.0 mL) were treated ac-
cording to the general procedure B. The crude product was purified by
silica gel column chromatography eluting with ethyl acetate/hexane
(2/1) and ethyl acetate/hexane (2/0.1) to afford the title compound 12
trile (ACN, 8.0 mL) were treated according to the general procedure A.
The crude product was purified by silica gel column chromatography
eluting with ethyl acetate/methanol (5/0.1) and triturated from acet-
on:diethyl ether to afford the title compound 9 as a white solid in 19%
1
yield. mp: 232.3 °C; H NMR (400 MHz, DMSO‑d
6
) δ: 1.23 (s, 3H, CH
3
2
),
),
1
1
.25 (s, 3H, CH
3
), 2.93–3.00 (m, 1H, CH), 3.46–3.50 (m, 2H, CH
as a white solid in 19% yield. mp: 174 °C; H NMR (400 MHz,
3
.65–3.70 (m, 2H, CH
2
), 7.30 (m, 2H, Ar-H, SO NH
2
2
), 7.39–7.36 (m,
DMSO‑d
2H, SO NH
(d, J = 8.7 Hz, 2H, Ar-H), 7.85 (t, J = 5.5 Hz, 1H, NHCSNH), 9.85 (s,
6
) δ: 3.55–3.57 (m, 2H, CH
2
), 3.90–3.94 (m, 2H, CH ), 7.30 (s,
2
4
H, Ar-H), 7.73 (d, J = 8.3 Hz, 2H, Ar-H), 7.80 (d, J = 8.7 Hz, 2H, Ar-
2
2
), 7.37 (d, J = 8.7 Hz, 2H, Ar-H), 7.41 (s, 4H, Ar-H), 7.81
1
3
H), 8.55–8.52 (m, 1H, CONH), 10.40 (brs, 1H, SO
2
NH). C NMR
1
3
(
100 MHz, DMSO‑d
6
) δ: 167.2 (C]O), 152.9, 142.1, 139.7, 132.5,
1H, NHCSNH), 10.41 (brs, 1H, SO
2
NH). C NMR (100 MHz, DMSO‑d )
6
1
28.2, 128.2 (2 X Ar-C), 127.1 (3 X Ar-C), 119.3 (2 X Ar-C), 51.1
δ: 181.4 (C]S), 142.1, 139.7, 138.6, 129.5 (3 X Ar-C), 129.3, 128.1 (2
(
eNHCH
2
CH
2
SO
2
−), 35.1 (eNHCH
2
CH
SO
2 2
−), 34.2, 24.5 (2 X CH
3
).
X Ar-C), 126.0, 119.4 (2 X Ar-C), 50.8 (eNHCH
2
CH
2
2
SO −), 39.5
−
1
−1
IR υmax (cm ): 3356, 3297 (NeH stretching), 1609 (amide I), 1538
(eNHCH
2
CH
2
SO −). IR υmax (cm ): 3368, 3267, 3213 (NeH
2
(
amide II), 1330 (SO
2
asymmetric stretching), 1145 (SO
2
symmetric
stretching), 1547 (NeH bending), 1319 (SO
2
asymmetric stretching),
−
stretching). m/z (ESI negative): 424.19 [M–H] . Elemental analysis
1227 (C]S stretching), 1140 (SO
2
symmetric stretching). m/z (ESI
−
calculated (%) for 0.2 C
3
H
6
O X C18
H
23
N
3
O
5
S
2
: C, 51.11; H, 5.58; N,
negative): 447.29 [M–H] . Elemental analysis calculated (%) for 0.4
9
.61; S, 14.67; found: C, 51.05; H, 5.32; N, 9.81; S, 14.46.
C
3
H
7
NO X C15
H
17ClN
4
O
4
S : C, 40.69; H, 4.17; N, 12.89; S, 20.11;
3
found: C, 40.88; H, 3.98; N, 12.70; S, 20.01.
4
.3.10. 4-(2-(3-(4-(tert-Buthyl)phenyl)thioureido)ethylsulfonamido)
benzenesulfonamide 10
4.3.13. 4-(2-(3-(4-Fluorophenyl)thioureido)ethylsulfonamido)
benzenesulfonamide 13
2
-(N-(4-sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, water
(
10 mL), 4-tert.buthylbenzoic acid (1.0 equiv.), 1-1′carbonyldiimida-
2-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, 4-
fluorophenyl isothiocyanate (1.0 equiv.), and diisopropylethylamine
(DIPEA, 2.0 equiv.), dry acetonitrile (ACN, 8.0 mL) were treated ac-
cording to the general procedure B. The crude product was purified by
silica gel column chromatography eluting with ethyl acetate/hexane
(2/1) and ethyl acetate/hexane (2/0.1) and triturated from
zole (1.0 equiv.), diisopropylethylamine (DIPEA, 2.0 equiv.), dry acet-
onitrile (ACN, 8.0 mL) were treated according to the general procedure
A. The crude product was purified by silica gel column chromatography
eluting with ethyl acetate/hexane (3/1) to afford the title compound 10
as a white solid in 17% yield. mp: 161.5 °C; 1H NMR (400 MHz,
6

Ö. Akgül, et al.
Bioorganic Chemistry 103 (2020) 104236
aceton:diethyl ether to afford the title compound 13 as a white solid in
(DIPEA, 2.0 equiv.), dry acetonitrile (ACN, 8.0 mL) were treated ac-
cording to the general procedure B. The crude product was purified by
silica gel column chromatography eluting with ethyl acetate/hexane/
ethanol (2/1/0.5) and crystallized from aceton:water to afford the title
1
1
9% yield. mp: 200.4 °C; H NMR (400 MHz, DMSO‑d
6
) δ: 3.52–3.56
(
(
m, 2H, CH
2
), 3.90–3.91 (m, 2H, CH
2
), 7.17–7.21 (m, 2H, Ar-H), 7.31
s, 2H, SO
2
NH ), 7.34–7.37 (m, 4H, Ar-H), 7.73–7.75 (s, 1H, NHCSNH),
2
1
7
.80 (d, J = 8.9 Hz, 2H, Ar-H), 9.76 (s, 1H, NHCSNH), 10.42 (brs, 1H,
compound 16 as a white solid in 21% yield. mp: 238.2 °C; H NMR
1
3
SO
2
NH). C NMR (100 MHz, DMSO‑d
6
) δ: 181.6 (C]S), 161.4, 158.9,
(400 MHz, DMSO‑d
6
) δ: 3.58–3.61 (m, 2H, CH ), 3.94–3.99 (m, 2H,
2
1
1
40.9 (d, J = 243 Hz, C4′), 135.7 (m, C2′), 128.1 (3 X Ar-C), 127.1,
27.01, 119.4 (2 X Ar-C), 116.3 (d, J = 22.17 Hz, C3′), 50.9
CH
2
), 7.31 (s, 2H, SO
2
NH ), 7.39 (d, J = 8.8 Hz, 2H, Ar-H), 7.81 (d,
2
J = 8.6 Hz, 4H, Ar-H), 8.21 (d, J = 9.2 Hz, 2H, Ar-H), 8.32–8.29
−
1
13
(
eNHCH
2
CH
2
2
SO −), 39.6 (eNHCH
2
CH
2
SO
2
−). IR υmax (cm ): 3371,
(m,1H, NHCSNH), 10.40 (s, 1H, NHCSNH), 10.45 (brs, 1H, SO
2
NH).
C
3
294, 3259 (NeH stretching) 1500 (NeH bending), 1315 (SO
2
asym-
NMR (100 MHz, DMSO‑d ) δ: 181.2 (C]S), 146.7, 143.2, 142.0, 139.8,
6
metric stretching), 1227 (C]S stretching), 1139 (SO
2
symmetric
128.1 (2 X Ar-C), 125.4 (2 X Ar-C), 122.0, 119.5 (3 X Ar-C), 50.5
−
−1
stretching). m/z (ESI negative): 431.29 [M–H] . Elemental analysis
(eNHCH
2
CH
2
SO
2
−), 39.5 (eNHCH
2
CH
2
2
SO ). IR υmax (cm ): 3384,
2
calculated (%) for 0.2 C
4
H
10O X C15
H
17FN
4
O
S
4 3
: C, 42.42; H, 4.28; N,
3340, 3240 (NeH stretching), 1543 (NO asymmetric stretching), 1512
1
2.53; S, 21.50; found: C, 42.62; H, 4.56; N, 12.52; S, 21.87.
(NeH bending), 1330 (NO
2
symmetric stretching), 1315, 1302 (SO
2
asymmetric stretching), 1265 (C]S stretching), 1146 (SO
2
symmetric
−
4
.3.14. 4-(2-(3-(4-Methoxyphenyl)thioureido)ethylsulfonamido)
stretching). m/z (ESI negative): 458.32 [M–H] . Elemental analysis
benzenesulfonamide 14
calculated (%) for 0.1 C
3
H
6
O X 0,1 H
2
O X C15
H
17
N
5
O
6
3
S : C, 39.34; H,
2
-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, 4-
3.84; N, 14.99; S, 20.59; Found: C, 39.17; H, 3.74; N, 15.27; S, 20.31.
methoxyphenyl isothiocyanate (1.0 equiv.), and diisopropylethylamine
(
DIPEA, 2.0 equiv.), dry acetonitrile (ACN, 8.0 mL) were treated ac-
4
.3.17. 4-(2-(3-(4-(Trifluoromethyl)phenyl)thioureido)ethylsulfonamido)
cording to the general procedure B. The crude product was purified by
silica gel column chromatography eluting with ethyl acetate/hexane
benzenesulfon amide 17
2
-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, 4-tri-
(
2/1) and crystallized from acetone/water to afford the title compound
fluoromethylphenyl isothiocyanate (1.0 equiv.), and diisopropylethy-
lamine (DIPEA, 2.0 equiv.), dry acetonitrile (ACN, 8.0 mL) were treated
according to the general procedure B. The crude product was purified
by silica gel column chromatography eluting with with ethyl acetate/
hexane (2/1) and ethyl acetate/hexane (2/0.1) and crystallized from
1
1
4 as a white solid in 30% yield. mp: 187 °C; H NMR (400 MHz,
DMSO‑d
m, 2H, CH
Ar-H), 7.30 (s, 2H, SO
m, 1H, NHCSNH), 7.80 (d, J = 8.7 Hz, 2H, Ar-H), 9.59 (s, 1H,
6
) δ: 3.51–3.54 (m, 2H, CH
), 6.93 (d, J = 9.1 Hz, 2H, Ar-H), 7.19 (d, J = 8.9 Hz, 2H,
NH ), 7.36 (d, J = 8.7 Hz, 2H, Ar-H), 7.53–7.55
2
), 3.78 (s, 3H, OCH
3
), 3.88–3.93
(
2
2
2
(
ethanol:water to afford the title compound 17 as a white solid in 39%
1
3
NHCSNH), 10.38 (brs, 1H, SO
81.5 (C]S), 157.8, 142.1, 139.7, 131.9, 128.1 (2 X Ar-C), 127.1 (2 X
Ar-C), 119.4 (2 Ar-C), 115.1 (2 Ar-C), 56.2, 51.1
eNHCH CH SO −), 39.6 (eNHCH CH SO
317, 3241, 3214 (NeH stretching), 1505 (NeH bending), 1344 (SO
2
NH). C NMR (100 MHz, DMSO‑d
6
) δ:
1
yield. mp: 199.5 °C; H NMR (400 MHz, DMSO‑d
6
) δ: 3.56–3.59 (m, 2H,
1
CH
2
), 3.92–3.97 (m, 2H, CH
2
), 7.30 (s, 2H, SO
2
NH ), 7.38 (d,
2
X
X
J = 8.7 Hz, 2H, Ar-H), 7.77 (s, 4H, Ar-H), 7.81 (d, J = 8.8 Hz, 2H, Ar-
−
1
(
2
2
2
2
2
2
−). IR υmax (cm ): 3369,
H), 8.07–8.09 (m, 1H, NHCSNH), 10.12 (s, 1H, NHCSNH), 10.42 (brs,
3
2
1
3
1
1
1
H, SO
2
NH). C NMR (100 MHz, DMSO‑d ) δ: 181.4 (C]S), 143.7,
6
asymmetric stretching), 1291 (OeC methoxyl stretching), 1219 (C]S
40.9 (q, J = 243 Hz, C-F3), 128.1 (2 X Ar-C), 126.7 (q, J = 4 Hz, C-4′),
stretching) 1149 (SO
2
symmetric stretching). m/z (ESI negative):
O X
26.6, 124.9, 124.7, 123.9, 123.4 (m, C-3′), 119.4 (2 X Ar-C), 50.7
−
4
43.33 [M–H] . Elemental analysis calculated (%) for 0.15 H
2
−
1
(
eNHCH
2
CH
2
SO
2
−), 39.5 (eNHCH
2
CH
2
SO −). IR υmax (cm ): 3342,
2
C
16
H
20
N
4
O
5
S : C, 42.97; H, 4.58; N, 12.53; S, 21.51; found: C, 42.62; H,
3
3
253 (NeH stretching), 1545 (NeH bending), 1317 (SO
2
asymmetric
4
.56; N, 12.52; S, 21.87.
stretching), 1230 (C]S stretching), 1140 (SO
2
symmetric stretching).
−
m/z (ESI negative): 481.38 [M–H] . Elemental analysis calculated (%)
4
.3.15. 4-(2-(3-(4-Methylphenyl)thioureido)ethylsulfonamido)
for C16
H
17
F
3
N
4
O
4
S : C, 39.83; H, 3.55; N, 11.61; S, 19.93; found: C,
3
benzenesulfonamide 15
3
9.69; H, 3.67; N, 11.41; S, 20.13.
2
-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, 4-me-
thylphenyl isothiocyanate (1.0 equiv.), and diisopropylethylamine
(
DIPEA, 2.0 equiv.), dry acetonitrile (ACN, 8.0 mL) were treated ac-
5. CA inhibition
An Applied Photophysics stopped-flow instrument was used for as-
cording to the general procedure B. The crude product was purified by
silica gel column chromatography eluting with with ethyl acetate/
hexane (3/1) and crystallized from acetone/water to afford the title
saying the CA catalyzed CO hydration activity [26]. Phenol red (at a
2
1
compound 15 as a white solid in 18% yield. mp: 162.7 °C; H NMR
concentration of 0.2 mM) was used as an indicator, working at the
absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) as a
(
400 MHz, DMSO‑d
6
) δ: 3.51–3.54 (m, 2H, CH
2
), 3.78 (s, 3H, OCH ),
3
3
.88–3.93 (m, 2H, CH
J = 8.9 Hz, 2H, Ar-H), 7.30 (s, 2H, SO
Ar-H), 7.53–7.55 (m, 1H, NHCSNH), 7.80 (d, J = 8.7 Hz, 2H, Ar-H),
2
), 6.93 (d, J = 9.1 Hz, 2H, Ar-H), 7.19 (d,
buffer, and 20 mM Na
2
SO (for maintaining constant ionic strength),
4
2
NH ), 7.36 (d, J = 8.7 Hz, 2H,
2
following the initial rates of the CA-catalyzed CO
2
hydration reaction
for a period of 10–100 s. The CO concentrations ranged from 1.7 to
2
1
3
9
.59 (s, 1H, NHCSNH), 10.38 (brs, 1H, SO
2
NH). C NMR (100 MHz,
17 mM for the determination of the kinetic parameters and inhibition
constants. For each inhibitor, at least six traces of the initial 5–10% of
the reaction were used for determining the initial velocity. The un-
catalyzed 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 distilled–deionized water and dilutions up to 0.01 nM
were done thereafter with the assay buffer. Inhibitor and enzyme so-
lutions were preincubated together for 15 min at room temperature
prior to the assay, to allow for the formation of the E–I complex. The
inhibition constants were obtained by non-linear least-squares methods
using PRISM 3 and the Cheng-Prusoffequation as reported earlier and
represent the mean from at least three different determinations. All CA
isoforms were recombinant proteins obtained inhouse, as reported
earlier [15,29–31].
DMSO‑d ) δ: 181.2 (C]S), 142.1, 139.7, 136.6, 135.0, 130.2 (2 X Ar-
6
C), 128.1 (2 X Ar-C), 124.8 (2 X Ar-C), 119.4 (2 X Ar-C), 51.0
(
(
eNHCH
2
CH
2
SO
2
−), 39.6 (eNHCH
2
CH
2
2
SO −), 21.4 (eCH ). IR υmax
3
−
1
cm ): 3371, 3259 (NeH stretching), 1549 (NeH bending), 1321 (SO
2
asymmetric stretching), 1239 (C]S stretching), 1141 (SO
2
symmetric
−
stretching). m/z (ESI negative): 427.30 [M–H] . Elemental analysis
calculated (%) for C16
H
20
4
N O
4
3
S : C, 44.84; H, 4.70; N, 13.07; S, 22.44;
found: C, 44.74; H, 4.724; N, 12.99; S, 22.11.
4
.3.16. 4-(2-(3-(4-Nitrophenyl)thioureido)ethylsulfonamido)
benzenesulfonamide 16
-(N-(4-Sulfamoylphenyl)sulfamoyl)ethanamine HCl salt D, 4-ni-
trophenyl isothiocyanate (1.0 equiv.), and diisopropylethylamine
2
7

Ö. Akgül, et al.
Bioorganic Chemistry 103 (2020) 104236
Declaration of Competing Interest
https://doi.org/10.1039/c0cc02707c.
[
[
[
15] C.L. Lomelino, B.P. Mahon, R. McKenna, F. Carta, C.T. Supuran, Kinetic and X-ray
crystallographic investigations on carbonic anhydrase isoforms I, II, IX and XII of a
thioureido analog of SLC-0111, Bioorganic Med. Chem. 24 (2016) 976–981,
https://doi.org/10.1016/j.bmc.2016.01.019.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
16] F. Pacchiano, F. Carta, P.C. McDonald, Y. Lou, D. Vullo, A. Scozzafava, S. Dedhar,
C.T. Supuran, Ureido-substituted benzenesulfonamides potently inhibit carbonic
anhydrase IX and show antimetastatic activity in a model of breast cancer metas-
tasis, J. Med. Chem. 54 (2011) 1896–1902, https://doi.org/10.1021/jm101541x.
17] M. Bozdag, F. Carta, M. Ceruso, M. Ferraroni, P.C. McDonald, S. Dedhar,
C.T. Supuran, Discovery of 4-hydroxy-3-(3-(phenylureido)benzenesulfonamides as
SLC-0111 analogues for the treatment of hypoxic tumors overexpressing carbonic
anhydrase IX, J. Med. Chem. 14 (2018) 6328–6338, https://doi.org/10.1021/acs.
jmedchem.8b00770.
Acknowledgements
This project was supported by The Scientific and Technical Research
Council of Turkey [TUBİTAK 116S705] and Ege University [BAP
2
018.BİL.002].
[
[
18] A. Scozzafava, L. Menabuoni, F. Mincione, F. Briganti, G. Mincione, C.T. Supuran,
Carbonic anhydrase inhibitors. Synthesis of water-soluble, topically effective, in-
traocular pressure-lowering aromatic/heterocyclic sulfonamides containing ca-
tionic or anionic moieties: is the tail more important than the ring? J. Med. Chem.
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