Quinoline-sulfamoyl carbamates/sulfamide derivatives: Synthesis, cytotoxicity, carbonic anhydrase activity, and molecular modelling studies

Article abstract of DOI:10.1016/j.bioorg.2021.104778

Carbonic anhydrase (CA) IX, and XII isoforms are known to be highly expressed in various human tissues and malignancies. CA IX is a prominent target for some cancers because it is overexpressed in hypoxic tumors and this overexpression leads to poor prognosis. Novel twenty-seven compounds in two series (sulfamoylcarbamate-based quinoline (2a-2o) and sulfamide-based quinoline (3a-3l)) were synthesized and characterized by means of IR, NMR, and mass spectra. Their inhibitory activities were evaluated against CA I, CA II, CA IX, and CA XII isoforms. 2-Phenylpropyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2m) exhibited the highest hCA IX inhibition with the K_i of 0.5 μM. In addition, cytotoxic effects of the synthesized compounds on human colorectal adenocarcinoma (HT-29; HTB-38), human breast adenocarcinoma (MCF7; HTB-22), human prostate adenocarcinoma (PC3; CRL-1435) and human healthy skin fibroblast (CCD-986Sk; CRL-1947) cell lines were examined. The cytotoxicity results showed that 2j, 3a, 3e, 3f are most active compounds in all cell lines (HT-29, MCF7, PC3, and CCD-986Sk).

Full text of DOI:10.1016/j.bioorg.2021.104778

Bioorganic Chemistry 110 (2021) 104778
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Quinoline-sulfamoyl carbamates/sulfamide derivatives: Synthesis,
cytotoxicity, carbonic anhydrase activity, and molecular modelling studies
,
Elmas Begum Cakmaka, Belma Zengin Kurtb *, Dilek Ozturk Civelekc, Andrea Angelid,
,
Atilla Akdemire, Fatih Sonmezf, Claudiu T. Supurand *, Mustafa Kucukislamoglug
a Sakarya University, Institute of Natural Sciences, 54050 Sakarya, Turkey
b Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34093 Istanbul, Turkey
c Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, 34093 Istanbul, Turkey
d
`
Universita degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
e Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Computer-aided Drug Discovery Laboratory, 34093 Istanbul, Turkey
f Sakarya University of Applied Sciences, Pamukova Vocational School, 54055 Sakarya, Turkey
g Sakarya University, Faculty of Arts and Science, Department of Chemistry, 54050 Sakarya, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
Carbonic anhydrase (CA) IX, and XII isoforms are known to be highly expressed in various human tissues and
malignancies. CA IX is a prominent target for some cancers because it is overexpressed in hypoxic tumors and this
overexpression leads to poor prognosis. Novel twenty-seven compounds in two series (sulfamoylcarbamate-based
quinoline (2a-2o) and sulfamide-based quinoline (3a-3l)) were synthesized and characterized by means of IR,
NMR, and mass spectra. Their inhibitory activities were evaluated against CA I, CA II, CA IX, and CA XII isoforms.
2-Phenylpropyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2m) exhibited the highest hCA IX inhibition with the Ki
of 0.5 µM. In addition, cytotoxic effects of the synthesized compounds on human colorectal adenocarcinoma (HT-
29; HTB-38), human breast adenocarcinoma (MCF7; HTB-22), human prostate adenocarcinoma (PC3; CRL-1435)
and human healthy skin fibroblast (CCD-986Sk; CRL-1947) cell lines were examined. The cytotoxicity results
showed that 2j, 3a, 3e, 3f are most active compounds in all cell lines (HT-29, MCF7, PC3, and CCD-986Sk).
Quinoline
Sulfamide
Sulfamoyl carbamate
CA inhibition
Cytotoxicity
Molecular docking
Molecular dynamics simulations
1. Introduction
bound isozymes of carbonic anhydrase (CA), namely CA IX and CA XII
[3]. CA IX is the most potent overexpressed gene in human cancer cells
Cancer is one the most common cause of death in the world. Multiple
mutations and epigenetic changes facilitate both the proliferation and
survival of tumor cells. The uncontrolled growth of malignant cells leads
to a high rate of nutrient and oxygen consumption. Inadequate envi-
ronments such as lack of oxygen (hypoxia) force tumor cells to initiate
response mechanisms that support cell life and migration. Therefore,
more than 70% of solid tumors have distinctively hypoxic micro-
environements [1]. The energy produced by glycolysis is important for
cancer cells to survive and multiply under hypoxic conditions. The in-
efficiency of glycolysis in ATP production causes an increase in glucose
consumption in hypoxic tumors. To make up for this deficiency, genes
encoding key proteins involved in angiogenesis (eg vascular endothelial
growth factor, VEGF), glucose transport (GLUT), glycolysis (eg phos-
phofructokinase 1, PFK-1), the export of monocarboxylic acids are
upregulated [2]. These genes include the genes encoding membrane-
in response to hypoxia [4]. CA IX catalyzes the reversible conversion of
carbon dioxide and thus plays a crucial role in stabilizing the extracel-
lular pH by protecting cancer cells from apoptosis under hypoxia. It has
been shown to have a similar overexpression profile for CA XII [5].
Physiologically, CA IX is present only with low expression in a small
group of normal tissues such as gastric mucosa epithelium, small in-
testine epithelium, and gall bladder. However, overexpression of CA IX
has been reported in many malignancies, including lung cancer, colon
cancer, breast cancer, cervical cancer, renal cell carcinoma, and bladder
cancer [6].
In general, carbonic anhydrases (CAs, EC 4.2.1.1) reversibly catalyze
the hydration of CO2 to bicarbonate and protons in all living organisms
[7]. The 15 different α-CA isoforms identified in humans (h) differ by
molecular features, some of such isoforms are cytosolic (CA I, CA II, CA
III, CA VII, and CA XIII), others are membrane-bound (CA IV, CA IX, CA
* Corresponding authors.
E-mail addresses: bzengin@bezmialem.edu.tr (B. Zengin Kurt), claudiu.supuran@unifi.it (C.T. Supuran).
https://doi.org/10.1016/j.bioorg.2021.104778
Received 28 October 2020; Received in revised form 15 January 2021; Accepted 21 February 2021
Available online 24 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

E.B. Cakmak et al.
Bioorganic Chemistry 110 (2021) 104778
XII, CA XIV, and CA XV), two of them are mitochondrial (CA VA and CA
VB), and one isozyme is secreted in saliva (CA VI) [8,9]. These CA iso-
forms have been shown to play an active role in the mechanism of many
diseases. Two cancer-associated CA isoforms IX and XII are actively
involved in carbon dioxide metabolism, thus playing a role in pH control
and tumor progression [5].
The first discovered molecules as CA inhibitors are the sulfonamides
and their isoesters. Many researches have shown that unlike sulfon-
amide compounds, various compounds such as dithiocarbamates, xan-
thates, phenolics, polyamines, and coumarins act as CA inhibitors
[10–13]. Recent studies show that (substituted) sulfamides, and sulfa-
moylcarbamates also exhibit some levels of CA inhibition [14–17]. The
sulfamide moiety is structurally similar to those of sulfonamide and
sulfamate, providing compounds bearing this structure to show a low
nanomolar affinity for the target enzymes for which they were designed.
This is achieved by the fact that the free or substituted sulfamide moiety
plays an important role in binding to the active site of the inhibitor. The
ability of the sulfamide group to give physicochemical properties similar
to drug molecules to the organic structures to which they are attached,
due to their properties such as increasing water solubility or showing
better bioavailability, provides an advantage to the sulfamide group
[18–20]. Quinoline is a skeleton that is widely used in medicinal
chemistry. Quinoline derivatives have various biological and pharma-
cological activities, such as antimalarial, antifungal, antibacterial,
antileishmanial and anticancer [21–25].
Scheme 2. Synthesis of new quinoline-sulfamide derivatives.
absorption at about 3200–3300 cmꢀ 1 relating to NH stretch of sulfamoyl
or sulfamide groups. The stretch of the carbamate carbonyl moiety
shows the absorbance between 1710 and 1740 cmꢀ 1. From the 1H NMR
spectra; for the 2a-2o; the signals for aromatic protons were observed
between 7.36 and 9.77 ppm and aliphatic protons recorded about at
0.75–5.05 ppm. For the 3a-3l; the signals for aromatic protons were
observed between 6.31 and 8.73 ppm, NH proton signals detected about
at 8.68–8.94 ppm, and aliphatic proton signals observed about at
0.59–5.73 ppm. From the 13C NMR spectra, the signals of the 149 and
156 ppm can be seen for carbonyl of carbamate groups. The signals of
the aliphatic and aromatic carbons were observed at 13–85 ppm and
114–154 ppm, respectively.
1H NMR, 13C NMR, and MS spectra of the synthesized compounds are
given in supplementary materials.
The approval of tumor-bound CA IX and XII, belonging to the family
of carbonic enzymes, as antitumor drug targets have created a good
research area for the synthesis of effective inhibitors of these enzymes.
This study aimed to investigate the synthesis of new quinoline de-
rivatives of bearing sulfamide/sulfamoylcarbamate and the biological
activities of these compounds, which may be effective inhibitors of CAs.
Their effects on CA I, CA II, CA IX, and CA XII isoforms, and cytotoxic
properties on the HT-29, MCF7, PC3, and CCD-986Sk cell lines were
evaluated. Furthermore, molecular modeling studies were performed to
suggest possible modes of binding of these inhibitors inside the active
sites of hCAs.
2.2. CA inhibition
The CA inhibitory profiles of the synthesized compounds were
evaluated by applying a stopped flow CO2 hydrase assay [29], using
acetazolamide (AAZ) as a standard inhibitor against four physiologically
significant isoforms CA I, II, IX, and XII.
From the Table 1, the Ki values of the sulfamoylcarbamate-based
quinolines (2a-2o) were determined in the range of 3.0 and > 100
µM, 0.6 and > 100 µM, 0.5 and > 100 µM, 6.9 and > 100 µM against hCA
I, hCA II, hCA IX and hCA XII, respectively. hCA XII was inhibited
weaker than other hCA isoforms (hCA I, hCA II, hCA IX). Among the first
series, compound 2k containing benzyl moiety showed the best inhibi-
tion of cytosolic isoform hCA I (associated with glaucoma) with Ki of 3.0
µM, which is close to that of AAZ (Ki of 0.25 µM) while compound 2g
bearing cycloheptyl moiety strongly inhibited the cytosolic isoform hCA
II with Ki of 0.6 µM. hCA IX and hCA XII (tumor-associated isoforms)
were inhibited by compound 2m including phenylpropyl moiety with Ki
of 0.5 µM and compound 2c bearing pentyl group with Ki of 6.9 µM,
respectively.
2. Result and discussion
2.1. Chemistry
In this study, two quinoline series (sulfamoylcarbamate-based
quinoline (2a-2o) and sulfamide-based quinoline (3a-3l) derivatives)
were synthesized.
The synthesis of first series consists of the reaction of various alcohols
with chlorosulfonyl isocyanate (1). The alcohols were mixed with 1 in
In addition, some notions about structure-activity relationship (SAR)
observed from Table 1: (i) The cyclization of pentyl group strongly
decreased the inhibitory activity against hCA XII (comparing 2c (Ki =
6.9 µM) with 2e (Ki = >100 µM)) and also the cyclization of hexyl group
reduced the inhibitory activity almost 14-fold against hCA II (comparing
2d (Ki = 5.5 µM) with 2f (Ki = 74.3 µM)).
◦
CH2Cl2 at 0 C [26] and added 8-amino quinoline with Et3N [27] to
obtain 2a-2o (Scheme 1).
The synthesis of second series consists of the reaction of
sulfamoylcarbamate-based quinoline with various amine derivatives.
The tert-butyl(N-(quinolin-8-yl)sulfamoyl)carbamate (2a) obtained by
above method used tert-butylalcohol was reacted with 1◦ or 2◦ amine
derivatives in THF at 60 ◦C [28] to give sulfamide-based quinoline (3a-
3l) derivatives (Scheme 2).
(ii) The presence of methylene between carbamate and t-butyl moi-
eties dramatically decreased the inhibitory activity against hCA I, II, IX
and XII (comparing 2a (Ki values of 27.3 µM, 75.6 µM, 0.7 µM, 8.5 µM
against hCA I, II, IX and XII, respectively) with 2b (Ki values of >100 µM
against all hCAs)). The similar effect was determined when compared 2l
(Ki values of 9.1 µM, 39.4 µM, 1.8 µM, 50.2 µM against hCA I, II, IX and
XII, respectively) with 2n (Ki values of >100 µM against all hCAs). In
contrast, the presence of methylene between carbamate and phenyl
raised the inhibitory activity against hCA I, II and XII (comparing 2j (Ki
values of 7.3 µM, 74.8 µM, and 81.5 µM, respectively) with 2k (Ki values
of 3.0 µM, 3.9 µM, and 22.5 µM, respectively)).
1
All of the newly synthesized compounds were characterized by H
NMR, 13C NMR, IR and MS. According to the IR spectra of the synthe-
sized 2a-2o and 3a-3l derivatives, it was possible to observe the
(iii) The methyl migration from β-position to α-position of carbamate
moiety extremely reduced the all hCA inhibition (comparing 2m (Ki
values of 6.3 µM, 2.2 µM, 0.5 µM, 39.05 µM against hCA I, II, IX and XII,
respectively) with 2n (Ki values of >100 µM against all hCAs)).
Scheme 1. Synthesis of new sulfamoylcarbamate-based quinoline derivatives.
Furthermore, the methyl or phenyl binding to α-position of carbamate
2

E.B. Cakmak et al.
Bioorganic Chemistry 110 (2021) 104778
Table 1
Table 2
In vitro inhibition Ki values of 3a-3l for the hCA I, II, IX and XII.
In vitro inhibition Ki values of 2a-2o for the hCA I, II, IX and XII.
Compound
R
Ki (µM)*
hCA I
Compound
-NRR’
Ki (µM)*
hCA I
hCA II
hCA IX
hCA XII
hCA II
hCA IX
hCA XII
2a
2b
27.3
75.6
0.7
8.5
3a
3b
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2c
2d
2e
3.7
3.3
6.5
7.2
5.5
5.8
0.6
0.6
0.7
6.9
>100
>100
3c
3d
>100
>100
>100
>100
>100
>100
>100
>100
2f
8.7
74.3
0.6
0.7
>100
>100
54.2
2g
2h
2i
6.3
0.6
3e
3f
>100
>100
>100
>100
5.8
70.8
>100
1.5
7.9
56.4
0.7
8.5
>100
>100
>100
3g
>100
>100
>100
>100
2j
7.3
74.8
0.8
81.5
3h
>100
>100
>100
>100
2k
2l
3.0
9.1
3.9
0.8
1.8
22.5
50.2
39.4
3i
3j
>100
>100
>100
>100
>100
>100
>100
>100
2m
2n
2o
6.3
2.2
0.5
39.0
>100
>100
>100
>100
>100
>100
>100
>100
3k
3l
>100
>100
>100
>100
>100
>100
>100
>100
AAZ
–
0.25
0.012
0.026
0.006
AAZ
–
0.25
0.012
0.026
0.006
*
Mean from 3 different assays, by a stopped flow technique.
*
Mean from 3 different assays, by a stopped flow technique.
IX and XII, respectively.
moiety also decreased hCA I, II, IX and XII inhibitions ranging from
almost 2-fold to 120-fold (comparing 2k (Ki values of 3.0 µM, 3.9 µM,
0.8 µM and 22.5 µM, respectively) with 2l (Ki values of 9.1 µM, 39.4 µM,
1.8 µM and 50.2 µM, respectively) and 2o (Ki values of >100 µM against
all hCAs)).
2.3. Cell viability against normal and cancer cell lines
Cell viability assay was carried out using human colorectal adeno-
carcinoma (HT-29; HTB-38), human breast adenocarcinoma (MCF7;
HTB-22), human prostate adenocarcinoma (PC3; CRL-1435) and human
healthy skin fibroblast (CCD-986Sk; CRL-1947) cell lines. Each cell line
was treated with the synthesized compounds for 48 h and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
was used to determine the viability of cells. Doxorubicin (dox) was used
as the reference compound. As shown in Table 3 2j, 3a, 3e, and 3f are
most active compounds against cancer cell lines. 3a, 3e and 3f are the
(iv) The ring expansion from cyclopentyl to cyclohexyl and cyclo-
heptyl did not show linear and appreciable effect on hCA I, IX and XII
inhibitions (comparing 2e (Ki values of 6.5 µM, 0.7 µM and >100 µM,
respectively) with 2f (Ki values of 8.7 µM, 0.7 µM and >100 µM,
respectively) and 2g (Ki values of 6.3 µM, 0.6 µM and >100 µM,
respectively)). On the other hand, hCA II inhibition was first decreased
almost 13-fold, and then increased almost 120-fold by the ring expan-
sion (comparing 2e (Ki = 5.8 µM) with 2f (Ki = 74.3 µM) and 2g (Ki =
0.6 µM)).
most active compounds with the IC50 values of 21.97
21.49 M, respectively, against HT-29 cell line. The most cytotoxic
compound against MCF7 cell line is 2j with the IC50 value of 12.75 M.
3a and 3f are the most cytotoxic compounds with the IC50 values of
14.77 M and 12.10 M, respectively, against PC3 cell line. The syn-
thesized compounds were also tested in healthy cell line CCD-986Sk. It
μM, 23.28 μM and
μ
From the Table 2, among the sulfamide-based compounds (3a-3l),
only 3f showed considerable inhibitory activity against all hCA isoforms.
The others weakly inhibited the all hCAs (Ki values of >100 µM). The Ki
values of the 3f are 7.9 µM, 56.4 µM, 0.7 µM and 8.5 µM against hCA I, II,
μ
μ
μ
3

E.B. Cakmak et al.
Bioorganic Chemistry 110 (2021) 104778
sidechain of Ser3. The substituents at position R1 points between the
sidechain of Gln92 and Pro202 and may form hydrohobic interactions
with the later residue. The R-isomer of the compounds adopts almost an
identical pose in which only the methyl group changes its orientation.
The ability of sulfamoylcarbamate series to adopt similar poses may
explain the relatively narrow range of Ki values that have been obtained.
A 50 ns MD simulation was performed on the hCA IX – 2m complex
that was obtained from the docking study. The RMSD values of the
binding pocket and protein (all heavy atoms) are generally below 2.5 Å
during the entire simulation (Fig. 1). The RMSD value of the ligand first
increases approximately to 6 Å and stays stable until 30 ns. After this
point, the RMSD value increases again. From the ligand-protein inter-
action scheme it becomes clear that after 30 ns of MD simulation com-
pound 2 m does not form any interaction with the pocket anymore
(Fig. 1E). During the first 30 ns of the simulation, compound 2m forms
frequently hydrogen bonds with the side chain of Trp5 and the backbone
of Pro201 (Fig. 1E). In addition, hydrogen bonds have been observed
with the sidechain (approximately 10–16 ns) and backbone (approxi-
mately 21–32 ns) of Gln2. Minor interaction are occassionaly observed
with Ser3 (hydrogen bonds and H-arene interactions) and very few with
Thr200 (hydrogen bonds) and Pro202 (H-arene interactions).
Table 3
IC50 values of the cytotoxicity of synthesized molecules at HT-29, MCF7, PC3,
and CCD cell line.
Compound
IC50
(μM)*
HT-29
MCF7
PC3
CCD
2a
2b
2c
2d
2e
2f
205.3 ± 35.34
42.80 ± 6.04
305.9 ± 53.77
308.0 ± 53.53
231.0 ± 34.67
174.0 ± 33.35
393.5 ± 60.39
285.1 ± 22.51
311.2 ± 56.01
29.15 ± 3.08
352.5 ± 93.08
230.0 ± 44.65
243.9 ± 39.37
325.4 ± 36.15
275.8 ± 47.13
21.97 ± 3.45
81.12 ± 15.56
46.59 ± 9.70
32.44 ± 6.86
23.28 ± 3.82
21.49 ± 3.06
41.82 ± 7.95
43.27 ± 10.42
37.50 ± 6.26
290.3 ± 100.3
57.38 ± 7.65
443.1 ± 95.47
14.51 ± 3.65
169.4 ± 27.09
27.37 ± 3.43
162.9 ± 14.22
191.1 ± 12.25
337.6 ± 53.06
269.7 ± 34.53
142.4 ± 14.04
116.1 ± 14.89
217.5 ± 42.43
12.75 ± 0.79
140.3 ± 10.95
112.8 ± 8.89
207.9 ± 29.69
155.6 ± 20.96
109.9 ± 11.39
19.82 ± 2.56
33.88 ± 5.26
37.33 ± 6.13
33.93 ± 3.92
19.81 ± 2.46
23.04 ± 3.56
50.13 ± 7.87
56.50 ± 10.41
23.83 ± 2.28
59.49 ± 6.77
45.82 ± 4.63
45.80 ± 4.34
0.46 ± 0.11
236.1 ± 30.84
25.92 ± 3.39
88.83 ± 12.42
145.1 ± 20.53
106.9 ± 10.40
127.7 ± 17.51
51.18 ± 6.59
115.0 ± 12.68
89.63 ± 10.89
43.21 ± 5.98
231.3 ± 37.79
81.44 ± 5.74
91.86 ± 8.08
60.92 ± 7.18
84.05 ± 7.86
14.77 ± 2.30
84.02 ± 11.2
33.77 ± 4.87
39.84 ± 5.76
21.74 ± 3.01
12.10 ± 1.70
26.58 ± 4.20
30.90 ± 6.19
22.49 ± 3.26
50.21 ± 7.60
38.76 ± 4.53
105.8 ± 8.16
1.85 ± 0.41
90.50 ± 15.13
22.39 ± 2.68
80.15 ± 13.02
118.7 ± 21.28
75.15 ± 13.49
85.58 ± 17.40
12.54 ± 1.75
61.31 ± 11.98
183.8 ± 41.12
32.31 ± 4.86
83.94 ± 14.80
69.54 ± 11.56
103.7 ± 22.11
68.99 ± 11.70
98.57 ± 19.39
15.09 ± 2.92
43.16 ± 7.51
27.56 ± 5.08
24.04 ± 5.92
13.23 ± 2.47
12.62 ± 2.13
32,36 ± 6,27
36.53 ± 8.04
19.69 ± 3.17
35.53 ± 6.20
36.28 ± 4.47
81.03 ± 20.35
0.16 ± 0.03
2g
2h
2i
2j
2k
2l
2m
2n
2o
3a
3b
3c
3d
3e
3f
Interestingly, the calculated energy of binding suggests that the
ligand binds with a similar energy between 6 and 50 ns of the MD
simulation. In the later stages of the simulation, the ligand’s sulfonamide
group approaches the positively charged N-terminal of the protein
(Glu0) and a hydrogen bond occurs. It should be noted that the hCA IX
crystal structure is a truncated protein without the N-terminal trans-
membrane domain. As such, the hydrogen bond between ligand and the
positively charged N-terminal of Glu0 is not considered of physiological
importance.
3g
3h
3i
3j
3k
3l
dox
*
The cell viability was represented as a percentage (%) relative to untreated
cells as a control.
The MD results suggest that the investigated hCA IX-2m complex
does not form strong bonds and that compound 2m may form multiple
binding poses with either the investigated active site or allosteric sites.
The obtained docked poses of sulfamoylcarbamate series in the
active site of hCA XII are more diverse compared to hCA IX. The poses
are scattered throughout the pocket and there is no common pose found
for the compounds. This may explain variation of measured Ki values.
The docked pose of compound 2c (Ki = 6.9 µM against hCA XII) is
shown in Fig. 2. Compound 2c is located between Thr91 and Ser132 and
forms hydrogen bonds with these residues. Again the sulfone group is
solvent exposed.
has been found that cytotoxicity results were almost similar against
CCD-986Sk as the cancer cell lines. 2g, 3a, 3e, and 3f showed cytotoxic
activity with the IC50 values of 12.54 μM, 15.09 μM, 13.23 μM and
12.62
μM, respectively, against CCD-986Sk cell line.
2.4. Molecular modelling studies
Docking studies in combination with molecular dynamics simula-
tions were conducted to suggest ligand-enzyme binding interactions for
the (sulfamoylcarbamate-based quinoline (2a-2o) and sulfamide-based
quinoline (3a-3l)) series with the active sites of the tumour-associated
hCA IX and XII and the widely distributed hCA I and II. Binding in-
teractions have been suggested for the sulfamoylcarbamates with the
lowest Ki value for hCA I, IX and XII. No stable binding interaction has
been obtained for the sulfamoylcarbamates in the hCA II active site.
Also, no docked poses could be suggested for sulfamide derivatives that
could explain the low Ki value for compound 3f versus the high Ki values
for the other members of the sulfamide series.
A 50 ns MD simulation shows that the docked pose of 2c changes
significantly, especially after 4 ns and stays at approximately 6 Å until
roughly 15 ns and then diminishes to 4 Å. At 20 ns the ligand RMSD
value increases again to approximately 10–14 Å (Fig. 2). Hydrogen
bonds are formed between the ligand in different poses and the side-
chains of Lys67 and Gln92. The GBVI/WSA ligand-protein binding en-
ergy increased from ꢀ 5 kcal/mol to approximately ꢀ 3/ꢀ 4 kcal/mol.
The MD simulation again suggests that 2c may bind to the hCA XII
active site with different poses, while allosteric sites cannot be excluded.
2.6. Docked poses of sulfamoylcarbamate series in the active sites of hCA
I
2.5. Suggested binding interactions of sulfamoylcarbamate series in the
active sites of the tumour-associated hCA IX and XII
The docked pose of compound 2k (Ki = 3.0 µM against hCA I) in the
active site of hCA I shows the presence of hydrogen bonds with the zinc-
bound water molecule, and the side chains of His64, His67 and Gln92
(Fig. 3). In addition, hydrophobic interactions are observed with the
sidechain of Phe91. All other compounds of sulfamoylcarbamates are
able to adopt similar poses.
Compound 2m shows the lowest measured Ki value among sulfa-
moylcarbamates (2a-2o) (Ki = 0.5 µM against hCA IX) with the excep-
tion of compounds 2b, 2i, 2n and 2o (Table 1). Nearly all compounds of
the sulfamoylcarbamate series adopt a similar docked pose as 2m (S-
isomer) in the hCA IX active site. The quinolone ring is located between
the sidechains of His94 and Leu198 and it may form H-arene in-
teractions with the zinc-bound water molecule (Fig. 1). Both ligand NH
functions could form hydrogen bonds with the backbone of Pro201 and
one of the oxygen atoms of the sulfone group forms a hydrogen bond
with the sidechain of Trp5, while the other oxygen atom is solvent
exposed. Carbonyl group of the ligand forms a hydrogen bond with the
During a 50 ns MD simulation the RMSD of the ligand increases
during the first nanosecond towards 4–5 Å, while the RMSD of the
pocket and protein is between 1 and 2 Å. The GBVI/WSA binding energy
changes from ꢀ 5 kcal/mol to approximately ꢀ 4 kcal/mol and fluctuates
around this value. The ligand-protein interactions after the ligand
reorientation in the first nanosecond stays stable and Hydrogen bonds
4

E.B. Cakmak et al.
Bioorganic Chemistry 110 (2021) 104778
Fig. 1. A) The docked pose of compound 2m in the active site of hCA IX. B) The pose after a 10 ns MD simulation. C) The RMSD values of ligand, pocket and protein.
D) The GBVI/WSA energy of binding. E) The ligand-protein interactions. Hydrogen bonds and interactions with the zinc ion are indicated in red dashed lines and H-
arene interactions are indicated in yellow dashed lines. The water molecule is indicated with a red sphere, while the zinc ion is indicated with a turquoise sphere. “a”:
backbone hydrogen bond acceptor interaction, “A”: sidechain hydrogen bond acceptor interaction, “R”: H-arene interaction and “d”: backbone hydrogen bond donor
interaction. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
are frequently formed with the sidechains of His67 and Gln92.
The MD simulations indicate that compound 2k, although flexible
and dynamic, is able to constantly form hydrogen bonds with the hCA I
active site.
quinoline (2a-2o) inhibited CA I, CA II, CA IX, and CA XII isoform at
micromolar levels (except for 2b, 2i, 2n, and 2o), while only 3f inhibited
all CA isoforms at the micromolar level, among the sulfamide-based
quinoline (3a-3l). These compounds were also evaluated in vitro on
HT-29 human colorectal adenocarcinoma, MCF-7 human breast
adenocarcinoma, PC3 human prostate adenocarcinoma, and CCD-986Sk
human healthy skin fibroblast cell lines. The effect of these derivatives
on four different cell lines varied according to the elements present in
their molecular structure. 3a and 3f showed high cytotoxicity on the HT-
29 cell line, whereas 2j showed a higher cytotoxic effect on MCF7 cell
line. Binding interactions between some of the hCAs have been inves-
tigated by computational methods, indicated that the possible ligand-
enzyme interactions for each of the enzymes. The results showed that
3. Conclusion
The synthesis of new quinolone sulfamoylcarbamate and sulfamide
derivatives and their inhibitory activity against four physiologically
relevant CA isoforms, CA I (cytosolic isoform), CA II (associated with
glaucoma and other pathologies), and CA IX and XII (tumor-associated
isoforms) were evaluated. The effect of these compounds on the four
different isoforms varied considerably. Sulfamoylcarbamates-based
5

E.B. Cakmak et al.
Bioorganic Chemistry 110 (2021) 104778
Fig. 2. (A) The docked pose of compound 2c in the active site of hCA XII. B) The pose after a 10 ns MD simulation. C) The RMSD values of ligand, pocket and protein.
D) The GBVI/WSA energy of binding. E) The ligand-protein interactions. Hydrogen bonds and interactions with the zinc ion are indicated in red dashed lines and H-
arene interactions are indicated in yellow dashed lines. The water molecule is indicated with a red sphere, while the zinc ion is indicated with a turquoise sphere. “a”:
backbone hydrogen bond acceptor interaction, “A”: sidechain hydrogen bond acceptor interaction, “R”: H-arene interaction and “D”: sidechain hydrogen bond donor
interaction. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the sulfamoylcarbamates-based quinoline series are interesting mole-
cules for CA inhibition and antiproliferative studies and most of them
are more active than sulfamide-based quinoline against hCA I, II, IX and
XII.
4. Experimental
4.1. Material and method
Melting points were taken on a STUART SMP40. IR spectra were
measured on Alfa Bruker spectrometer.1H and 13C NMR spectra were
measured on a Bruker spectrometer at 500 and at 125 Hz, respectively.
1H and 13C chemical shifts are referenced to the internal deuterated
6

E.B. Cakmak et al.
Bioorganic Chemistry 110 (2021) 104778
Fig. 3. A) The docked pose of compound 2k in the active site of hCA I. B) The pose after a 10 ns MD simulation. C) The RMSD values of ligand, pocket and protein. D)
The GBVI/WSA energy of binding. E) The ligand-protein interactions. Hydrogen bonds and interactions with the zinc ion are indicated in red dashed lines and H-
arene interactions are indicated in yellow dashed lines. The water molecule is indicated with a red sphere, while the zinc ion is indicated with a turquoise sphere. “A”:
sidechain hydrogen bond acceptor interaction, “R”: H-arene interaction and “D”: sidechain hydrogen bond donor interaction. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
solvent. Mass spectra were obtained using Thermo Fisher Scientific LC-
HRMS spectrometer. Spectrophotometric analyses were performed by a
BioTek Power Wave XS (BioTek, USA). The cell line was purchased from
American Type Culture Collection (ATCC). Dulbecco’s Modified Eagle’s
Medium-F12, RPMI Medium, fetal calf serum, and PBS were purchased
from GIBCO BRL, InVitrogen (Carlsbad, CA). The chemicals and solvents
were purchased from Fluka Chemie, Merck, Alfa Aesar and Sigma-
Aldrich.
4.2. General procedures and spectral data
Synthesis of compounds 2a-2o: Chlorosulfonyl isocyanate (1) (1.2
mmol) and alcohol derivatives (1.2 mmol) were dissolved in dichloro-
methane (DCM) (5 mL), and the solution was stirred at 0 ◦C until 30 min
[26]. It was then carefully added to a solution of 8-aminoquinoline (1.2
mmol) and triethylamine (1.2 mmol) in DCM at 0 ◦C and was stirred for
1 h. The mixture was concentrated in vacuo, and the residue was dis-
solved in EtOAc and washed with water and brine. The organic layer was
7

E.B. Cakmak et al.
Bioorganic Chemistry 110 (2021) 104778
dried over MgSO4 and concentrated under reduced pressure to give 2a-
2o [27]. The crude products were purified by column chromatography
on silica gel from Hexan: Ethylacetate.
758, 726, 609 cmꢀ 1; 1H NMR (DMSO‑d6, 500 MHz) δ/ppm: 8.93 (dd, J
= 4.2, 1.5 Hz, 1H), 8.46 (dd, J = 8.3, 1.5 Hz, 1H), 7.76 (dd, J = 8.1, 1.1
Hz, 1H), 7.75–7.62 (m, 3H), 4.71–4.49 (m, 1H), 1.60–1.48 (m, 2H),
1.44–1.29 (m, 7H), 1.17–1.27 (m 3H); 13C NMR (CDCl3, 125 MHz)
δ/ppm: 150.1, 148.9, 138.3, 136.3, 133.1, 128.2, 126.9, 122.7, 122.2,
115.0, 79.0, 33.2, 28.1, 22.4; HRMS (ESI): C17H21N3O4S [M+Na]+;
calculated for 363.13, found 386.1143.
tert-butyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2a): Whitish
powder, 92% yield, mp. 120–121 ◦C; IR: 3133, 2990, 1714, 1509, 1476,
1369, 1327, 1150, 1058, 924, 826, 762, 589 cmꢀ 1; 1H NMR (CDCl3, 500
MHz) δ/ppm: 8.85 (dd, J = 4.22, 1.64 Hz, 1H), 8.19 (dd, J = 8.29, 1.62
Hz, 1H), 7.82 (dd, J = 7.51, 1.30 Hz, 1H), 7.59 (dd, J = 8.29, 1.27 Hz,
1H), 7.54 (d, J = 7.59 Hz, 1H), 7.52–7.46 (m, 1H), 1.28 (s, 9H) 13C NMR
(CDCl3, 125 MHz) δ/ppm: 149.0, 148.9, 138.4, 136.3, 133.2, 128.2,
127.0, 122.8, 122.1, 115.4, 84.3, 27.6; HRMS (ESI): C14H17N3O4S
[M+Na]+; calculated for 323.09, found 346,0830.
adamantan-1-yl (N-(quinolin-8-yl)sulfamoyl)carbamate (2h) Yellow
powder, 28% yield, mp. 237–238 ◦C; IR: 3172, 2906, 2853, 1739, 1503,
1450, 1410, 1373, 1317, 1228, 1148, 1086, 1035, 953, 868, 793, 759,
718, 628 cmꢀ 1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.80 (dd, J = 4.2, 1.6
Hz, 1H), 8.13 (dd, J = 8.2, 1.5 Hz, 1H), 7.76 (dd, J = 7.5, 1.2 Hz, 1H),
7.53 (dd, J = 8.28, 1.22 Hz, 1H), 7.48 (d, J = 7.61 Hz, 1H), 7.47–7.40
(m, 1H), 2.04–1.99 (m, 3H), 1.84 (d, J = 2.63 Hz, 6H), 1.52–1.46 (q, J =
12.50, 12.50, 12.50 Hz, 6H); 13C NMR (CDCl3, 125 MHz) δ/ppm: 148.8,
148.6, 138.4, 136.3, 133.3, 128.2, 127.0, 122.7, 122.1, 115.3, 84.2,
40.7, 35.8, 30.8; HRMS (ESI): C20H23N3O4S [M+Na]+; calculated for
401.14, found 424, 1298.
neopentyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2b) White pow-
der, 23% yield, mp. 108–109 ◦C; IR: 3200, 2956, 1743, 1503, 1453,
1414, 1381, 1316, 1235, 1157, 1087, 1058, 937, 878, 825, 793, 772,
758 cmꢀ 1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.75 (d, J = 3.24 Hz, 1H),
8.10 (d, J = 8.15 Hz, 1H), 7.74 (d, J = 7.38 Hz, 1H), 7.50 (d, J = 8.04 Hz,
1H), 7.46 (d, J = 7.71 Hz, 1H), 7.44–7.39 (m, 1H), 3.65 (m, 2H), 0.76 (s,
9H) 13C NMR (CDCl3, 125 MHz) δ/ppm: 156.4, 154.6, 142.9, 141.9,
138.2, 133.2, 131.9, 128.0, 127.8, 120.1, 79.5, 36.5, 30.8; HRMS (ESI):
2-isopropyl-5-methylcyclohexyl (N-(quinolin-8-yl)sulfamoyl)carba-
mate (2i) Yellow powder, 45% yield, mp. 155, 156 ◦C; IR: 3226, 3158,
2950, 1699, 1505, 1464, 1414, 1390, 1349, 1247, 1160, 1087, 932, 874,
825, 778, 767 cmꢀ 1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.63 (dd, J =
4.2, 1.6 Hz, 1H), 7.96 (dd, J = 8.3, 1.5 Hz, 1H), 7.57 (dd, J = 7.4, 1.2 Hz,
1H), 7.35 (dd, J = 8.2, 1.2 Hz, 1H), 7.31 (d, J = 7.5 Hz, 1H), 7.29–7.25
(m, 1H), 4.23 (dt, J = 10.9, 4.4 Hz, 1H), 1.42–1.28 (m, 3H), 1.22–1.17
(m, 1H), 1.13–0.93 (m, 2H), 0.72–0.59 (m, 1H), 0.51 (d, J = 6.7 Hz, 3H),
0.49–0.41 (m, 1H), 0.38 (d, J = 6.9 Hz, 3H), 0.13 (d, J = 6.93 Hz, 3H);
13C NMR (CDCl3, 125 MHz) δ/ppm: 150.0, 149.0, 138.2, 136.2, 133.2,
128.2, 126.8, 122.6, 122.2, 114.7, 46.8, 40.1, 33.8, 31.1, 25.8, 23.0,
21.8, 20.5, 15.7; HRMS (ESI): C20H27N3O4S [M+Na]+; calculated for
405.17, found 428.1611.
C
15H19N3O4S [M+Na]+; calculated for 337.11, found 360.0986.
pentyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2c) Whitish powder,
37% yield, mp. 90–91 ◦C; IR: 3211, 3952, 2931, 1725, 1505, 1443,
1416, 1378, 1348, 1311, 1238, 1162, 1087, 929, 885, 825, 810, 795,
760, 657, 641 cmꢀ 1; 1H NMR (DMSO‑d6, 500 MHz) δ/ppm: 8.93 (d, J =
4.09 Hz, 1H), 8.46 (d, J = 8.34 Hz, 1H), 7.76 (d, J = 8.15 Hz, 1H),
7.70–7.62 (m, 3H), 3.92 (t, J = 6.68 Hz, 2H), 1.24–1.12 (m, 2H),
0.95–0.80 (m, 2H), 0.64–0.62 (m, 5H); 13C NMR (CDCl3, 125 MHz)
δ/ppm: 150.5, 148.9, 138.3, 136.3, 133.1, 128.2, 126.9, 122.7, 122.2,
114.9, 65.7, 36.9, 24.6, 22.5, 22.3; HRMS (ESI): C15H19N3O4S
[M+Na]+; calculated for 337.11, found 360.0987.
hexyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2d) Yellow powder,
26% yield, mp. 119–120 ◦C; IR: 3230, 2930, 2854, 1728, 1504, 1451,
1415, 1375, 1343, 1240, 1113, 1088, 941, 872, 848, 826, 795, 793, 630
cmꢀ 1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.78 (d, J = 4.19 Hz, 1H), 8.13
(d, J = 8.27 Hz, 1H), 7.75 (d, J = 7.49 Hz, 1H), 7.53 (d, J = 8.02 Hz, 1H),
7.48 (d, J = 7.68 Hz, 1H), 7.46–7.42 (m, 1H), 3.97 (t, J = 6.75 Hz, 2H),
1.58–1.52 (m, 2H), 1.46–1.09 (m, 6H), 0.78–0.75 (t, J = 6.92 Hz, 3H);
13C NMR (CDCl3, 125 MHz) δ/ppm: 150.5, 148.9, 138.4, 136.2, 133.1,
128.2, 126.8, 122.7, 122.2, 114.9, 67.3, 31.2, 28.2, 25.1, 22.4, 13.9;
HRMS (ESI): C16H21N3O4S [M+Na]+; calculated for 351.13, found
374.1143.
phenyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2j) White powder,
31% yield, mp. 176–177 ◦C; IR: 3284, 3220, 1738, 1666, 1536, 1503,
1491, 1470, 1319, 1163, 945 cmꢀ 1
;
1H NMR (DMSO‑d6, 500 MHz)
δ/ppm: 9.77 (s, 1H) 8.92 (dd, J = 4.2, 1.6 Hz, 1H), 8.87 (dd, J = 4.2, 1.6
Hz, 1H), 8.40 (dd, J = 8.3, 1.5 Hz, 1H), 8.39–8.34 (m, 1H), 8.25 (dd, J =
7.7, 1.1 Hz, 1H), 7.79 (dd, J = 7.5, 1.2 Hz, 1H), 7.71 (dd, J = 8.3, 1.1 Hz,
1H), 7.67–7.57 (m, 3H), 7.48 (d, J = 9.5, 6.4 Hz, 1H); 13C NMR
(DMSO‑d6, 125 MHz) δ/ppm: 156.4, 149.9, 148.4, 138.2, 137.0, 134.4,
133.6, 127.3, 123.1, 122.7, 122.2, 119.5, 115.5, 114.6; HRMS (ESI):
C
16H13N3O4S [M+H2O+MeOH]+; calculated for 393.41, found
393.2972.
cyclopentyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2e) Whitish
powder, 35% yield, mp. 129–130 ◦C; IR: 3239, 2957, 2872, 1720, 1504,
1455, 1434, 1415, 1384, 1342, 1312, 1236, 1154, 1087, 926, 893, 827,
795, 773 cmꢀ 1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.77 (dd, J = 4.22,
1.61 Hz, 1H), 8.12 (dd, J = 8.29, 1.57 Hz, 1H), 7.75 (dd, J = 7.34, 1.44
Hz, 1H), 7.49 (dd, J = 8.27, 1.42 Hz, 1H), 7.46 (d, J = 7.40 Hz, 1H),
7.44–7.40 (m, 1H), 5.05–4.85 (m, 1H), 1.64–1.60 (m, 2H), 1.58–1.51
(m, 4H), 1.45–1.42 (m, 2H); 13C NMR (CDCl3, 125 MHz) δ/ppm: 150.2,
148.9, 138.3, 136.3, 133.1, 128.2, 126.9, 122.7, 122.2, 114.9, 80.8,
32.3, 23.4; HRMS (ESI): C15H17N3O4S [M+Na]+; calculated for 335,
found 358.0829.
benzyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2k) Yellow powder,
64% yield, mp. 90–91 ◦C; IR: 3211, 2953, 2631, 2867,1725, 1504, 1442,
1416, 1376, 1347, 1312, 1237, 1162, 1133, 1086, 930, 884, 795, 760,
657 cmꢀ 1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.69 (dd, J = 4.21, 1.48
Hz, 1H), 8.14 (dd, J = 8.29, 1.42 Hz, 1H), 7.75 (dd, J = 7.56, 1.05 Hz,
1H), 7.53 (dd, J = 8.27, 1.03 Hz, 1H), 7.47 (d, J = 7.79 Hz, 1H),
7.46–7.39 (m, 1H), 7.12–7.07 (m, 3H), 7.18–7.15 (m, 2H), 4.98 (s, 2H);
13C NMR (CDCl3, 125 MHz) δ/ppm: 150.5, 148.9, 138.3, 136.3, 133.1,
128.2, 126.9, 122.7, 122.2, 114.9, 65.7; HRMS (ESI): C17H15N3O4S
[M+H]+; calculated for 360.10, found 360.0987.
1-phenylethyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2l) White
powder, 25% yield, mp. 110–111 ◦C IR: 3243, 3217, 1737, 1594, 1504,
1450, 1436, 1414, 1383, 1344, 1312, 1232, 1162, 1135, 1087, 938, 836,
796, 754, 732, 698 cmꢀ 1; 1H NMR (DMSO‑d6, 500 MHz) δ/ppm: 8.86
(dd, J = 4.22, 1.61 Hz, 1H), 8.45 (dd, J = 8.33, 1.55 Hz, 1H), 7.77 (dd, J
= 8.15, 1.16 Hz, 1H), 7.70 (dd, J = 7.61, 1.27 Hz, 1H), 7.66–7.62 (m,
2H), 7.20–7.12 (m, 3H), 7.11–7.05 (m, 2H), 5.59 (q, J = 6.5 Hz, 1H),
1.29 (d, J = 6.6 Hz, 3H); 13C NMR (DMSO‑d6, 125 MHz) δ/ppm: 150.8,
149.9, 141.2, 137.3, 133.4, 128.6, 128.4, 127.8, 127.2, 126.0, 125.8,
cyclohexyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2f) Yellow
powder, 71% yield, mp. 122–123 ◦C; IR: 3293, 3260, 2931, 2851, 1747,
1505, 1431, 1381, 1315, 1223, 1150, 1006, 952, 930, 872, 751, 609
cmꢀ 1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.76 (dd, J = 4.2, 1.6 Hz, 1H),
8.11 (dd, J = 8.2, 1.5 Hz, 1H), 7.74 (dd, J = 7.50, 1.23 Hz, 1H), 7.50 (dd,
J = 8.27, 1.16 Hz, 1H), 7.49–7.37 (m, 2H), 4.66–4.36 (m, 1H),
1.64–1.47 (m, 4H), 1.11–1.24 m, 6H); 13C NMR (CDCl3, 125 MHz)
δ/ppm: 150.0, 148.9, 138.3, 136.3, 133.1, 128.2, 126.9, 122.7, 122.2,
115.0, 31.0, 25.1, 23.2; HRMS (ESI): C16H19N3O4S [M+Na]+; calculated
for 349.11, found 372.0985.
123.4, 123.0, 115.5, 74.4, 22.2; HRMS (ESI):
C18H17N3O4S
C
17H15N3O4S [M+Na]+; calculated for 371.09, found 394.0831.
cycloheptyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2g) Whitish
powder, 27% yield, mp. 144–145 ◦C; IR: 3247, 3170, 2924, 2859, 1705,
1504, 1459, 1414, 1374, 1311, 1236, 1168, 1085, 928, 864, 823, 792,
2-phenylpropyl (N-(quinolin-8-yl)sulfamoyl)carbamate (2m) White
powder, 22% yield, mp. 111–112 ◦C IR: 3252, 2972, 2958, 2923, 1740,
1506, 1470, 1453, 1437, 1416, 1375, 1338, 1242, 1162, 926, 880, 825,
8

E.B. Cakmak et al.
Bioorganic Chemistry 110 (2021) 104778
793, 757, 697 cmꢀ 1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.76 (dd, J =
4.08, 1.62 Hz, 1H), 8.11 (dd, J = 8.2, 1.5 Hz, 1H), 7.72 (dd, J = 7.56,
1.04 Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H), 7.4–7.04
(m, 1H), 7.14 (t, J = 7.30 Hz, 2H), 7.11–7.06 (m, 1H), 6.99 (d, J = 7.14
Hz, 2H), 4.03 (d, J = 7.49 Hz, 2H), 2.93–2.85 (m, 1H), 1.08 (d, J = 7.02
Hz, 3H); 13C NMR (CDCl3, 125 MHz) δ/ppm: 150.3, 149.0, 143.2, 142.2,
138.4, 136.2, 133.0, 128.5, 128.2, 127.3, 127.1, 126.8, 122.8, 122.2,
115.1, 71.6, 38.7, 17.5; HRMS (ESI): C19H19N3O4S C17H15N3O4S
[M+Na]+; calculated for 385.11, found 408.0986.
1,1-diisopropyl-N-(N-(quinolin-8-yl)sulfamide (3c) Grey powder,
40% yield, mp. 106–107 ◦C; IR: 3293, 2976, 2934, 1592, 1470, 1364,
1334, 1301, 1174, 1133, 1066, 1020, 966, 916, 879, 789, 673 cmꢀ 1; 1H
NMR (CDCl3, 125 MHz) δ/ppm: 8.88 (s, 1H), 8.69 (dd, J = 4.22, 1.64 Hz,
1H), 8.05 (dd, J = 8.28, 1.65 Hz, 1H), 7.76 (dd, J = 8.89, 4.45 Hz, 1H),
7.40–7.37 (m, 2H), 7.35–7.31 (m, 1H), 3.63–3.57 (m, 2H), 1.09 (d, J =
7.03 Hz, 12H); 13C NMR (CDCl3, 125 MHz) δ/ppm: 148.6, 138.5, 136.3,
134.5, 128.1, 126.8, 121.8, 121.5, 115.0, 48.8, 21.6; HRMS (ESI):
C15H21N3O2S [M+Na]+; calculated for 307.1354, found 330.1242.
butyl(N-(quinolin-8-yl)sulfamide (3d) Grey liquid, 38% yiled; IR:
3275, 2959, 2935, 2871, 1580, 1469, 1432, 1410, 1329, 1301, 1124,
1084, 1057, 917, 822, 790, 754, 653 cmꢀ 1; 1H NMR (CDCl3, 500 MHz)
δ/ppm: 8.94 (s, 1H), 8.73 (dd, J = 4.10, 1.33 Hz, 1H), 8.09 (dd, J = 8.27,
1.31 Hz, 1H), 7.68 (t, J = 4.40, 4.40 Hz, 1H), 7.44 (d, J = 4.43 Hz, 2H),
7.40 (t, J = 8.26, 3.98, 3.98 Hz, 1H), 4.73 (s, J = 5.55, 5.55 Hz, 1H), 2.94
(t, J = 20.11, 6.76, 6.76 Hz, 2H), 1.31–1.20 (m, 2H), 1.11–1.00 (m, 2H),
0.62–0.59 (t, J = 7.37, 7.37 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δ/ppm:
148.7, 138.3, 136.3, 134.3, 128.2, 127.0, 122.0, 121.6, 114.2, 42.9,
31.1, 19.5, 13.4; HRMS (ESI): C13H17N3O2S [M+Na]+; calculated for
279.1041, found 302.0933.
1-phenylpropan-2-yl (N-(quinolin-8-yl)sulfamoyl)carbamate (2n)
White powder, 32% yield, mp. 107–108 ◦C; IR: 3272, 3209, 1721, 1508,
1442, 1415, 1379, 1312, 1242, 1150, 1090, 925, 884, 823, 756, 741,
705, 665, 650 cmꢀ 1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.74 (dd, J =
4.22, 1.60 Hz, 1H), 8.10 (dd, J = 8.30, 1.55 Hz, 1H), 7.73 (dd, J = 7.55,
1.18 Hz, 1H), 7.51 (dd, J = 8.27, 1.07 Hz, 1H), 7.44 (t, J = 7.7 Hz, 1H),
7.40 (dd, J = 8.28, 4.23 Hz, 1H), 7.09–7.00 (m, 3H), 6.95–6.87 (m, 2H),
4.89–4.83 (m, 1H), 2.69 (dd, J = 13.6, 6.0 Hz, 1H), 2.52 (dd, J = 13.66,
6.8 Hz, 1H), 0.95 (d, J = 6.31 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
δ/ppm: 156.6, 149.9, 149.0, 138.3, 136.3, 133.1, 129.5, 129.3, 128.3,
126.9, 126.6, 122.7, 122.2, 115.0, 75.1, 41.6, 18.8; HRMS (ESI):
C
19H19N3O4S [M+Na]+; calculated for 385.11, found 408.0986.
benzhydryl (N-(quinolin-8-yl)sulfamoyl)carbamate (2o) Grey pow-
N-(quinolin-8-yl)pyrrolidine-1-sulfamide (3e) Brown powder, 43%
◦
yield, mp. 80–81 C; IR: 3445, 3276, 2974, 1721, 1681, 1505, 1471,
der, 20% yield, mp. 138–139 ◦C; IR: 3251, 3096, 3033, 1730, 1483,
1505, 1471, 1437, 1367, 1334, 1305, 1195, 1151, 1055, 1011, 913, 824,
792, 754 cmꢀ 1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.87 (s, 1H), 8.73
(dd, J = 4.19, 1.61 Hz, 1H), 8.09 (dd, J = 8.28, 1.62 Hz, 1H), 7.71 (dd, J
= 5.51, 3.36 Hz, 1H), 7.47–7.41 (m, 2H), 7.39 (dd, J = 8.27, 4.19 Hz,
1H), 3.29–3.27 (m, 4H), 1.69–1.66 (m, 4H); 13C NMR (CDCl3, 125 MHz)
δ/ppm: 148.6, 138.3, 136.3, 134.7, 128.2, 126.9, 121.9, 121.4, 114.7,
48.4, 25.5; HRMS (ESI): C13H15N3O2S [M+Na]+; calculated for
277.0885, found 300.0777.
1455, 1416, 1386, 1228, 1159, 1090, 925, 878, 861, 769, 763, 744, 697,
610 cmꢀ 1; H NMR (DMSO‑d6, 500 MHz) δ/ppm: 8.75 (dd, J = 4.21,
1
1.59 Hz, 1H), 8.44 (dd, J = 8.33, 1.56 Hz, 1H), 7.77 (dd, J = 8.16, 1.05
Hz, 1H), 7.70 (dd, J = 7.61, 1.21 Hz, 1H), 7.66–7.58 (m, 2H), 7.35 (d, J
= 4.36 Hz, 1H), 7.19–7.17 (m, 5H), 7.16–7.13 (m, 4H), 6.60 (s, 1H); 13
C
NMR (DMSO‑d6, 125 MHz) δ/ppm: 150.6, 149.8, 140.0, 138.2, 137.1,
133.3, 128.8, 128.4, 128.2, 127.1, 126.9, 126.6, 123.3, 123.0, 115.4,
78.6; HRMS (ESI): C23H19N3O4S [M+Na]+; calculated for 433.11, found
456.0986.
N-(quinolin-8-yl)piperidine-1-sulfamide (3f) Pink powder, 25%
yield, mp. 128–129 ◦C; IR: 3245, 2943, 2846, 1581, 1411, 1373, 1339,
Synthesis of compounds 3a-3l: Chlorosulfonyl isocyanate (1) (1.2
mmol) and t-BuOH (1.2 mmol) were dissolved in dichloromethane
1308, 1278, 1159, 1089, 1054, 933, 920, 845, 823, 795, 766, 716 cmꢀ 1
;
1H NMR (CDCl3, 500 MHz) δ/ppm: 8.84 (s, 1H), 8.74 (dd, J = 4.19, 1.62
Hz, 1H), 8.14–8.06 (m, 1H), 7.70 (dd, J = 5.32, 3.56 Hz, 1H), 7.46–7.41
(m, 2H), 7.40 (dd, J = 8.25, 4.21 Hz, 1H), 3.21–3.15 (m, 4H), 1.44 (dd, J
= 11.19, 5.51 Hz, 4H), 1.34 (dd, J = 9.60, 4.16 Hz, 2H); 13C NMR
(CDCl3, 125 MHz) δ/ppm: 148.6, 138.3, 136.3, 134.7, 128.2, 126.9,
121.9, 121.3, 114.8, 47.2, 25.2, 23.5; HRMS (ESI): C14H17N3O2S
[M+Na]+; calculated for 291.1041, found 314.0931.
◦
(DCM) (5 mL), and the solution was stirred at 0 C until 30 min. The
mixture was then carefully added to a solution of 8-aminoquinoline (1.2
mmol) and triethylamine (1.2 mmol) in DCM at 0 ◦C and stirred for 1 h.
The mixture was concentrated in vacuo, and the residue was dissolved in
EtOAc and washed with water and brine. The organic layer was dried
over MgSO4 and concentrated under reduced pressure to give 2a. 2a
(1.25 mmol), amine derivatives (1.25 mmol), and Et3N (1.25 mmol) in
THF were solved and stirred at 60 ◦C, 18 h. The solution was concen-
trated in vacuo, and the residue was dissolved in EtOAc and washed with
water and brine. The organic layer was dried over MgSO4 and concen-
trated under reduced pressure to give 3a-3l [28]. The crude products
were purified by column chromatography on silica gel from Hexan:
Ethylacetate.
cyclohexyl (N-(quinolin-8-yl)sulfamide) (3g) White powder, 33%
yield, mp. 133–134 ◦C; IR: 3274, 2935, 2857, 1504, 1470, 1414, 1363,
1305, 1164, 1058, 931, 885, 823, 794, 754, 657 cmꢀ 1; 1H NMR (CDCl3,
500 MHz) δ/ppm: 8.75 (s, 1H), 8.70–8.63 (m, 1H), 8.11–7.96 (m, 1H),
7.56 (dd, J = 6.23, 2.32 Hz, 1H), 7.34–7.26 (m, 3H), 6.52 (s, 1H), 3.09
(s, 1H), 2.92 (s, 1H), 1.54–1.10 (m, 4H), 1.09–0.90 (m, 6H); 13C NMR
(CDCl3, 125 MHz) δ/ppm: 148.4, 138.0, 136.2, 134.6, 128.0, 126.8,
121.8, 120.9, 113.7, 52.7, 39.9, 33.4, 24.7; HRMS (ESI): C15H19N3O2S
[M+Na]+; calculated for 305.1198, found 328.1089.
1,1-diethyl-N-(N-(quinolin-8-yl)sulfamide (3a) Grey powder, 30%
◦
yield, mp. 86–87 C; IR: 3308, 2977, 2931, 2882, 1621, 1582, 1470,
1410, 1361, 1334, 1306, 1142, 1014, 924, 821, 785, 759, 687 cmꢀ 1; 1H
NMR (CDCl3, 500 MHz) δ/ppm: 8.84 (s, 1H), 8.66 (dd, J = 4.23, 1.66 Hz,
1H), 8.02 (dd, J = 8.29, 1.67 Hz, 1H), 7.62–7.52 (m, 1H), 7.42–7.34 (m,
2H), 7.36–7.29 (m, 1H), 3.21 (q, J = 7.17 Hz, 4H), 0.94–0.91 (t, J =
7.21 Hz, 6H); 13C NMR (CDCl3, 125 MHz) δ/ppm: 148.6, 138.3, 136.3,
134.5, 128.1, 126.8, 122.0, 121.4, 114.5, 42.0, 13.4; HRMS (ESI):
C13H17N3O2S [M+Na]+; calculated for 279.1041, found 302.0932.
propyl (quinolin-8-yl)sulfamide (3b) White powder, 34% yield, mp.
100–101 ◦C; IR: 3297, 2966, 2935, 2875, 1579, 1503, 1460, 1409, 1361,
1351, 1333, 1308, 1148, 1131, 1066, 1006, 931, 817, 789, 747 cmꢀ 1; 1H
NMR (CDCl3, 500 MHz) δ/ppm: 8.94 (s, 1H), 8.73 (dd, J = 4.20, 1.62 Hz,
1H), 8.09 (dd, J = 8.28, 1.63 Hz, 1H), 7.68 (dd, J = 8.88, 4.47 Hz, 1H),
7.44 (d, J = 4.36 Hz, 2H), 7.40 (dd, J = 8.28, 4.21 Hz, 1H), 4.78 (s, 1H),
2.90 (q, J = 6.89 Hz, 2H), 1.37–1.27 (m, 2H), 0.65 (t, J = 7.41 Hz, 3H);
13C NMR (CDCl3, 125 MHz) δ/ppm: 147.6, 137.2, 135.3, 133.2, 127.1,
125.9, 121.0, 120.5, 113.1, 43.9, 21.4, 9.9; HRMS (ESI): C12H15N3O2S
[M+Na]+; calculated for 265.0885, found 288.0775.
2-(cyclohex-1-en-1-yl)ethyl-(N-(quinolin-8-yl)sulfamide) (3h) White
powder, 35% yield, mp. 87–88 ◦C; IR: 3306, 3266, 2932, 1503, 1472,
1415, 1326, 1306, 1137, 1082, 1064, 1025, 961, 921, 865, 823, 792,
759 cm-1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.94 (s, 1H), 8.75 (dd, J =
4.21, 1.64 Hz, 1H), 8.10 (dd, J = 8.29, 1.64 Hz, 1H), 7.68 (dd, J = 6.32,
2.52 Hz, 1H), 7.45 (d, J = 1.99 Hz, 2H), 7.41 (dd, J = 8.23, 4.17 Hz, 1H),
5.14 (s, 1H), 4.54 (t, J = 5.46 Hz, 1H), 3.02–2.93 (m, 2H), 1.89 (t, J =
6.29, 6.29 Hz, 2H), 1.79–1.72 (m, 2H), 1.34 (t, J = 6.63, 6.63 Hz, 2H),
1.29–1.22 (m, 4H); 13C NMR (CDCl3, 125 MHz) δ/ppm: 148.8, 138.3,
136.3, 134.3, 133.2, 128.2, 127.0, 124.8, 122.1, 121.7, 114.3, 40.2,
36.8, 27.0, 25.053, 22.4, 22.0; HRMS (ESI): C17H21N3O2S [M+Na]+;
calculated for 331.1354, found 354.1244.
benzyl(N-(quinolin-8-yl)sulfamide (3i) White powder, 47% yield,
◦
mp. 85–86 C; IR: 3443, 3265, 2930, 1679, 1504, 1472, 1454, 1412,
1365, 1332, 1305, 1147, 1087, 1051, 925, 825, 791 cmꢀ 1
;
1H NMR
(CDCl3, 500 MHz) δ/ppm: 8.68 (s, 1H), 8.08 (dd, J = 8.3, 1.6 Hz, 1H),
7.67 (dd, J = 6.8, 1.9 Hz, 1H), 7.44–7.41 (m, 2H), 7.39–7.36 (m, 1H),
9

E.B. Cakmak et al.
Bioorganic Chemistry 110 (2021) 104778
7.01–6. 99 (m, 3H), 6.93–6.91 (m, 2H), 4.07 (d, J = 5.3 Hz, 2H); 13C
NMR (CDCl3, 125 MHz) δ/ppm: 148.7, 138.3, 136.2, 135.9, 134.1,
128.5, 128.2, 127.8, 127.8, 126.9, 122.0, 121.7, 114.4, 47.3; HRMS
All necessary hCA crystal structures were obtained from the Broo-
khaven Protein Data Bank, i.e. hCA I in complex with topiramate (pdb:
3lxe, 1.9 Å), hCA II in complex with 2,5-dihydroxybenzoic acid and a
zinc-bound water molecule (pdb: 4e3d, 1.6 Å), hCA IX (pdb: 3iai, 2.2 Å)
and hCA XII (pdb: 1jd0, 1.5 Å) both in complex with acetazolamide. The
zinc-bound water molecule of the hCA II structure and all ligands
(acetazolamide, topiramate and 2,5-dihydroxybenzoic acid) were
retained and all other non-protein atoms were deleted from the crystal
structures. Only chain A was retained if more than one protein structure
was present. Hydrogen atoms were added with the “protonate 3D” [35]
tool of the MOE software package and subsequently a steepest-descent
energy minimization was performed using the AMBER14:EHT force
field. The four protein structures were superposed on the backbone
(ESI):
C
16H15N3O2S [M+Na]+; calculated for 313.0885, found
336.0775.
4-benzyl-N-(quinolin-8-yl)piperidine-1-sulfamide (3j) White pow-
der, 29% yield, mp. 105–106 ◦C; IR: 3255, 2921, 1504, 1469, 1455,
1415, 1381, 1341, 1311, 1241, 1162, 1087, 1062, 1044, 926, 823, 791,
743, 719, 699, 640 cmꢀ 1; 1H NMR (CDCl3, 500 MHz) δ/ppm: 8.82 (s,
1H), 8.70 (dd, J = 4.21, 1.64 Hz, 1H), 8.06 (dd, J = 8.29, 1.63 Hz, 1H),
7.69 (dd, J = 6.68, 2.19 Hz, 1H), 7.40–7.37 (m, 2H), 7.14 (dd, J = 11.89,
4.76 Hz, 2H), 7.12–7.04 (m, 1H), 6.93 (dd, J = 13.77, 6.78 Hz, 2H), 3.74
(d, J = 12.27 Hz, 2H), 2.57–2.52 (m, 2H), 2.31 (d, J = 7.24 Hz, 2H),
1.50–1.47 (m, 2H), 1.40–1.29 (m, 2H), 1.06–1.03 (m, 1H); 13C NMR
(CDCl3, 125 MHz) δ/ppm: 148.7, 139.8, 138.3, 136.3, 134.6, 129.0,
128.3, 128.2, 126.9, 126.0, 122.0, 121.5, 115.0, 46.7, 42.6, 37.3, 31.3;
HRMS (ESI): C21H23N3O2S [M+Na]+; calculated for 381.1511, found
404.1401.
atoms of hCA I (Cα atoms, RMSD: 1.395 Å, for 236 residues). The co-
ordinates of the hCA II zinc-bound water molecule were copied into the
other hCA structures.
Docking studies were performed using the FlexX docking tool
(v2.3.2; BioSolveIT GmbH, St. Augustin, Germany) within MOE. The
binding pocket was described as all residues within 11 Å of the coc-
rystallized ligand. The highest scoring three poses were retained and
analysed for complementarity between ligand and binding pocket with
respect to sterics, interactions and electrostatics.
benzo[d][1,3]dioxol-5-yl-methyl(N-(quinolin-8-yl)sulfamide) (3k)
Whitish powder, 37% yield, mp. 140–141 ◦C; IR: 3361, 3266, 1505,
1492, 1445, 1411, 1379, 1363, 1303, 1250, 1149, 1042, 925, 825, 757
1
cmꢀ 1; H NMR (CDCl3, 500 MHz) δ/ppm: 8.89 (s, 1H), 8.71 (dd, J =
4.19, 1.61 Hz, 1H), 8.10 (dd, J = 8.29, 1.60 Hz, 1H), 7.66 (dd, J = 6.37,
2.45 Hz, 1H), 7.51–7.42 (m, 2H), 7.42–7.35 (m, 1H), 6.40 (t, J = 7.9 Hz,
2H), 6.36 (d, J = 1.24 Hz, 1H), 5.73 (s, 2H), 4.00 (d, J = 5.87 Hz, 2H);
13C NMR (CDCl3, 125 MHz) δ/ppm: 148.6, 147.6, 147.1, 138.2, 136.2,
134.1, 129.5, 128.2, 126.9, 122.0, 121.7, 121.4, 114.3, 108.3, 108.0,
101.0, 47.2; HRMS (ESI): C17H15N3O4S [M+Na]+; calculated for
357.0783, found 380.0674.
4.6. Molecular dynamics simulations
In this study, the Yasara Structure software package (v18.8.9,
YASARA Biosciences GmbH) with the PME method was used for mo-
lecular dynamics simulations [36–38]. In the system prepared, the
steepest-descent protocol (AMBER14) was used to minimize the energy
[39,40]. The system was executed according to the whole procedure and
all bonds were restricted using LINCS and SETTLE algorithms [41,42].
Snapshots were taken every 100 ps of the 10 ns production run. The
binding energy (MM/PBSA) was calculated with Yasara Structure, while
the RMSD values as well as the binding interactions (protein–ligand
interaction fingerprint) were calculated with MOE.
3,4-dimethoxyphenethyl(N-(quinolin-8-yl)sulfamide) (3l) Whitish
powder, 38% yield, mp. 116–117 ◦C; IR: 3328, 3292, 1592, 1579, 1504,
1458, 1438, 1409, 1353, 1332, 1314, 1236, 1146, 1102, 1024, 950, 913,
1
875, 819, 781 cmꢀ 1; H NMR (CDCl3, 500 MHz) δ/ppm: 8.93 (s, 1H),
8.75 (dd, J = 4.20, 1.59 Hz, 1H), 8.11 (dd, J = 8.28, 1.55 Hz, 1H), 7.53
(dd, J = 7.54, 1.13 Hz, 1H), 7.44–7.40 (m, 2H), 7.39–7.32 (m, 1H), 6.57
(d, J = 8.08 Hz, 1H), 6.43–6.31 (m, 2H), 4.50 (s, 1H), 3.76 (s, 3H), 3.62
(s, 3H), 3.19 (s, 2H, br), 2.56 (t, J = 6.68 Hz, 2H); 13C NMR (CDCl3, 125
MHz) δ/ppm: 149.0, 148.7, 147.7, 138.2, 136.3, 134.1, 129.8, 128.2,
126.9, 122.0, 121.6, 120.5, 114.1, 111.4, 111.1, 55.8, 55.7, 44.2, 34.8;
HRMS (ESI): C19H21N3O4S [M+Na]+; calculated for 387.1253, found
410.1143.
Declaration of Competing Interest
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.
4.3. CA inhibition assays
Acknowledgments
In order to determine the CA inhibition of the compounds, the
method mentioned in the previous studies was used and inhibition re-
sults were obtained as described in refs. [29–34].
This work was supported by the Bezmialem Research Fund of the
Bezmialem Vakif University. Project Number: 4.2019/11.
Appendix A. Supplementary data
4.4. Cell cytotoxicity assay
1H and 13C NMR and MS spectra of the synthesized compounds and
dose curve graphs of enzyme inhibition and controls are given in Sup-
plementary Materials. Supplementary data to this article can be found
online at https://doi.org/10.1016/j.bioorg.2021.104778.
The cytotoxicity of the test compounds on human colorectal adeno-
carcinoma cell line (HT-29; HTB-38), human breast adenocarcinoma cell
line (MCF7; HTB-22), human prostate adenocarcinoma cell line (PC3;
CRL-1435) and human healthy skin fibroblast cell line (CCD-986Sk;
CRL-1947) were evaluated by MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay according to described methods
[8].
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