Synthesis, biological activity and multiscale molecular modeling studies for coumaryl-carboxamide derivatives as selective carbonic anhydrase IX inhibitors

Article abstract of DOI:10.1080/14756366.2017.1354857

New coumaryl-carboxamide derivatives with the thiourea moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and their inhibitory activity against the human carbonic anhydrase (hCA) isoforms hCA I, II, VII and IX were evaluated. While the hCA I, II and VII isoforms were not inhibited by the investigated compounds, the tumour-associated isoform hCA IX was inhibited in the high nanomolar range. 2-Oxo-N-((2-(pyrrolidin-1-yl)ethyl)carbamothioyl)-2H-chromene-3-carboxamide (e11) exhibited a selective inhibitory action against hCA IX with the K_i of 107.9 nM. In order to better understand the inhibitory profiles of studied molecules, multiscale molecular modeling approaches were used. Different molecular docking algorithms were used to investigate binding poses and predicted binding energies of studied compounds at the active sites of the CA I, II, VII and IX isoforms.

Full text of DOI:10.1080/14756366.2017.1354857

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis, biological activity and multiscale
molecular modeling studies for coumaryl-
carboxamide derivatives as selective carbonic
anhydrase IX inhibitors
Belma Zengin Kurt, Fatih Sonmez, Serdar Durdagi, Busecan Aksoydan,
Ramin Ekhteiari Salmas, Andrea Angeli, Mustafa Kucukislamoglu & Claudiu
T. Supuran
To cite this article: Belma Zengin Kurt, Fatih Sonmez, Serdar Durdagi, Busecan Aksoydan,
Ramin Ekhteiari Salmas, Andrea Angeli, Mustafa Kucukislamoglu & Claudiu T. Supuran (2017)
Synthesis, biological activity and multiscale molecular modeling studies for coumaryl-carboxamide
derivatives as selective carbonic anhydrase IX inhibitors, Journal of Enzyme Inhibition and
Medicinal Chemistry, 32:1, 1042-1052, DOI: 10.1080/14756366.2017.1354857
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1354857
© 2017 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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Date: 27 August 2017, At: 18:19

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 1042–1052
https://doi.org/10.1080/14756366.2017.1354857
RESEARCH PAPER
Synthesis, biological activity and multiscale molecular modeling studies for
coumaryl-carboxamide derivatives as selective carbonic anhydrase IX inhibitors
Belma Zengin Kurta, Fatih Sonmezb, Serdar Durdagic, Busecan Aksoydanc, Ramin Ekhteiari Salmasc, Andrea Angelid,
Mustafa Kucukislamoglub and Claudiu T. Supurand
b
aFaculty of Pharmacy, Department of Pharmaceutical Chemistry, Bezmialem Vakıf University, Istanbul, Turkey; Faculty of Arts and Science,
c
Department of Chemistry, Sakarya University, Sakarya, Turkey; Computational Biology and Molecular Simulations Laboratory, Department of
d
Biophysics, School of Medicine, Bahcesehir University, Istanbul, Turkey; Dipartimento Neurofarba, Sezione di ScienzeFarmaceutiche e
ꢀ
Nutraceutiche, Universita degli Studi di Firenze, Florence, Italy
ABSTRACT
ARTICLE HISTORY
Received 22 May 2017
Revised 9 July 2017
Accepted 10 July 2017
New coumaryl-carboxamide derivatives with the thiourea moiety as a linker between the alkyl chains
and/or the heterocycle nucleus were synthesized and their inhibitory activity against the human carbonic
anhydrase (hCA) isoforms hCA I, II, VII and IX were evaluated. While the hCA I, II and VII isoforms were not
inhibited by the investigated compounds, the tumour-associated isoform hCA IX was inhibited in the
high nanomolar range. 2-Oxo-N-((2-(pyrrolidin-1-yl)ethyl)carbamothioyl)-2H-chromene-3-carboxamide (e11)
exhibited a selective inhibitory action against hCA IX with the Ki of 107.9 nM. In order to better understand
the inhibitory profiles of studied molecules, multiscale molecular modeling approaches were used.
Different molecular docking algorithms were used to investigate binding poses and predicted binding
energies of studied compounds at the active sites of the CA I, II, VII and IX isoforms.
KEYWORDS
Coumarin; carboxamid;
thiourea; carbonic
anhydrase; molecular
docking; induced fit
docking; quantum polarised
ligand docking
specifically bind hypoxic tumour cells expressing this isoform17–24
Therefore, it has been considered that the CA inhibitors are crucial
molecules for the synthesis of new-generation anticancer drugs25.
Coumarin is one of the most known class of CA inhibitors
(CAIs) and shows their effect as 2-hydroxycinnamic acid hydrolysis,
unlike other inhibitors26,27. Coumarin derivatives show high select-
ivity to inhibit isoforms, especially in pharmacological applications,
such as the tumour-associated ones (hCA IX and XII, which are tar-
gets for antitumour/antimetastatic drugs) or the mitochondrial
.
1. Introduction
The carbonic anhydrases (CAs; EC 4.2.1.1) are a superfamily of met-
alloenzymes that present in all organisms and consist of metallic
core of Zn2þ ion at their active center1–4. CA, explored in the beef
erythrocytes for the first time, reversibly catalyses the reactions of
hydration of CO2 and dehydration5 of HCO3ꢀ. Many CA isozymes
involved in these processes are important therapeutic targets with
the potential to be inhibited/activated for the treatment of a
range of disorders such as oedema, glaucoma, obesity, cancer, epi-
lepsy, amyloid beta, leukaemia and osteoporosis6–10. However, the
physiologically relevant reaction that CAs catalyse, using as sub-
strates CO2, COS, CS2, cyanamide, carboxylic, phosphoric and thio-
carboxylicesters11–14. Sixteen different a-CA isoforms were isolated
from mammals, where they play crucial physiological roles. Some
of them are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are
membrane-bound (CA IV, CA IX, CA XII, CA XIV and CA XV), CA VA
and CA VB are mitochondrial, and CA VI is secreted in saliva and
milk11. Recent studies suggested that the necrosis formed around
a tumour depends on both the excessively expressing of CA IX
ones (CA VA and VB, which are targets for antiobesity agents)28,29
Thiocoumarin, thioxocoumarin and sulphocoumarin derivatives
showed high affinity for CA IX-XII even at low concentrations30–32
.
.
Urea and thiourea compounds work as building block in the
synthesis of heterocyclic compounds. These compounds, thanks to
their pharmacological properties, make a significant contribution
in the field of medicinal chemistry. Urea and thiourea derivatives
exhibit many biological activities such as analgesic, anti-inflamma-
tory, antimicrobial and anticancer. Thiourea derivatives are valu-
able building blocks for the synthesis of amides, guanidines and
varieties of heterocycles33,34. It has been reported that compounds
containing urea or thiourea as well as sulphonamide groups highly
enzymes increased at such domain and the controlling of pH15,16
.
Especially, hCA IX is expressed in a restricted number of normal
tissues, whereas it is over expressed in many solid tumours and
considered involved in important processes connected with cancer
progression. The over expression of hCA IX induces the pH imbal-
ance of tumour tissue contributing significantly to the extracellular
acidification of solid tumour; thereby hCA IX inhibitors could
inhibit the enzyme carbonic anhydrase35–37
.
Continuing our interest in coumarin CAIs, in this work, we
report the synthesis of novel thiourea-substituted coumaryl-car-
boxamid derivatives and their effects on the inhibitory activity of
human carbonic anhydrase hCA I, hCA II, hCA VII and hCA IX.
CONTACT Serdar Durdagi
Medicine, Bahcesehir University, Istanbul, Turkey; Fatih Sonmez
University, Sakarya, Turkey
serdar.durdagi@med.bau.edu.tr
Computational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of
fsonmez@sakarya.edu.tr Faculty of Arts and Science, Department of Chemistry, Sakarya
Supplemental data for this article can be accessed here
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1043
2.2.5. 2-oxo-N-(propylcarbamothioyl)-2H-chromene-3-carboxamide
(e2)
2. Experimental
2.1. Material and method
Yellow powder, 58% yield; mp. 123 ꢁC; IR: 3339, 3053, 2964, 1703,
1655, 1607, 1518, 1450, 1362, 1158, 755 cmꢀ1 1H NMR (CDCl3,
;
Melting points were taken on a Barnstead Electrothermal 9200. IR
spectra were measured on a Shimadzu Prestige-21 (200 VCE) spec-
trometer. 1H and 13C NMR spectra were measured on a Varian
300 MHz) d/ppm: 1.00 (3H, t, J ¼ 7.6 Hz), 1.60–1.72 (2H, m),
3.40–4.47 (2H, m), 7.35–7.42 (2H, m), 7.64–7.71 (2H, m), 8.83 (1H, s,
NH), 8.92 (1H, s); 13C NMR (CDCl3, 75 MHz) d/ppm: 11.7, 22.9, 41.8,
116.8, 118.7, 118.9, 125.5, 130.0, 134.1, 148.4, 154.6, 161.6, 161.7.
LC-MS (m/z): 321.2 [Mþ]. Anal. Calcd. for C14H14N2O3S; C, 57.92; H,
4.86; N, 9.65; found: C, 57.90; H, 4.85; N, 9.67.
1
Infinity Plus spectrometer at 300 and at 75 Hz, respectively. H and
13C chemical shifts are referenced to the internal deuterated solv-
ent. Mass spectra were obtained using MICROMASS Quattro LC-
MS-MS spectrometer. The elemental analyses were carried out
with a Leco CHNS-932 instrument. Spectrophotometric analyses
were performed by a BioTek Power Wave XS (Winooski, VT). The
chemicals and solvents were purchased from Fluka Chemie
(Taufkirchen, Germany), Merck (Taufkirchen, Germany), Alfa Aesar
(Taufkirchen, Germany) and Sigma-Aldrich (Taufkirchen, Germany).
2.2.6. N-(diethylcarbamothioyl)-2-oxo-2H-chromene-3-carboxamide
(e3)
Yellow powder, 40% yield; mp. 143 ꢁC; IR: 3300, 3060, 2978, 1709,
1609, 1567, 1437, 1219, 1198, 1016, 763, 641 cmꢀ1 1H NMR
;
(CDCl3, 300 MHz) d/ppm: 1.35 (5H, s, br), 1.64 (1H, s), 3.63 (2H, s,
br), 4.01 (2H, s, br), 7.27–7.46 (2H, m), 7.70–7.75 (2H, m), 8.96 (1H,
s), 10.97 (N¼C–SH, 1H, s, SH); 13C NMR (CDCl3, 75 MHz) d/ppm:
11.5, 47.7, 117.0, 118.0, 118.6, 123.4, 125.9, 130.4, 135.2, 150.5,
154.8, 158.5, 161.7, 177.7. LC-MS (m/z): 321.1 [Mþ]. Anal. Calcd. for
C15H16N2O3S; C, 59.19; H, 5.30; N, 9.20; found: C, 59.17; H, 5.31; N,
9.22.
2.2. General procedures of synthesis and spectral data
2.2.1. 2-oxo-2H-chromene-3-carboxylic acid (c)
A mixture of benzaldehyde (a) (3 mmol), meldrum’s acid (b)
(4.5 mmol) was stirred at reflux for 10 h. The mixture was cooled,
filtered and recrystallised from methanol to get product (c).
Spectral data of this compound were matched with the literature,
white solid, 91% yield; mp. 145–147 ꢁC38.
2.2.7. N-(diisopropylcarbamothioyl)-2-oxo-2H-chromene-3-carboxa-
mide (e4)
2.2.2. 2-oxo-2H-chromene-3-carbonyl chloride (d)
Yellow powder, 70% yield; mp. 150–151 ꢁC; IR: 3231, 3047, 2978,
A 2-oxo-2H-chromene-3-carboxylic acid (c) (0.01 mol) and SOCl2
(0.05 mol) were taken in round bottom flask and it was stirred for
4 h at 80 ꢁC temperature. After the excess SOCl2 was evaporated,
the crude product was purified by ether. Spectral data of this com-
1713, 1672, 1608, 1499, 1333, 1200, 1107, 757, 663 cmꢀ1 1H NMR
;
(CDCl3, 300 MHz) d/ppm: 1.23–1.58 (14H, m), 7.21–7.45 (2H, m),
7.71–7.75 (2H, m), 8.98 (1H, s), 10.68 (N¼C-SH, 1H, s, SH); 13C NMR
(CDCl3, 75 MHz) d/ppm:, 21.0, 46.4, 51.8, 117.0, 118.1, 118.7, 125.0,
125.9, 130.4, 135.1, 150.5, 154.8, 161.7, 164.1. LC-MS (m/z): 333.3
[Mþ]. Anal. Calcd. for C17H20N2O3S; C, 61.42; H, 6.06; N, 8.43; found:
C, 61.40; H, 6.07; N, 8.41.
pound was matched with the literature50
.
White solid, 99% yield; IR: 3057, 1737, 1676, 1605, 1557, 1417,
1225, 1179, 1040, 761 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d/ppm:
;
7.36–7.43 (2H, m), 7.69 (1H, td, J ¼ 1.7, 7.3 Hz), 7.87 (1H, dd, J ¼ 1.4,
7.6 Hz), 8.71 (1H, s); 13C NMR (DMSO-d6, 75 MHz) d/ppm:, 116.7,
118.6, 118.9, 125.4, 130.8, 134.9, 149.1, 155.1, 157.3, 164.5.
2.2.8. N-(cyclohexylcarbamothioyl)-2-oxo-2H-chromene-3-carboxa-
mide (e5)
2.2.3. N-(R-carbamothioyl)-2-oxo-2H-chromene-3-carboxamide
(e1–20)
Yellow powder, 53% yield; mp. 164–166 ꢁC; IR: 3321, 3051, 2925,
1704, 1665, 1609, 1523, 1452, 1366, 1163, 761, 610 cmꢀ1 1H NMR
;
A mixture of 2-oxo-2H-chromene-3-carbonyl chloride (1 mmol) and
KSCN (1,2 mmol) in CH3CN (30 ml) was heated under reflux for
30 min. Then, 1.2 mmol amine derivatives were added in the mix-
ture and the solution was refluxed for 4 h. The solution was evapo-
rated and the residue was extracted with water/CH2Cl2. The
organic phase was washed by water for three times and dried
over Na2SO4. After the organic solvent was evaporated, the crude
product was recrystallised from methanol to get pure crystalline
e1–20 in 25–70% yields.
(CDCl3, 300 MHz) d/ppm: 1.24–1.49 (4H, s), 1.61–1.65 (2H, m),
1.73–1.78 (2H, m), 1.97–2.00 (2H, m), 3.94–4.00 (1H, m), 7.35–7.42
(2H, m), 7.63–7.71 (2H, m), 8.76 (1H, d, J ¼ 6.7 Hz, NH), 8.9 (1H, s);
13C NMR (CDCl3, 75 MHz) d/ppm:, 24.9, 25.8, 32.9, 48.7, 116.8,
118.9, 125.4, 129.9, 134.1, 148.3, 150.9, 154.6, 160.6, 161.7. LC-MS
(m/z): 330.2 [Mþ]. Anal. Calcd. for C17H18N2O3S; C, 61.80; H, 5.49; N,
8.48; found: C, 61.81; H, 5.47; N, 8.49.
2.2.9.
2-oxo-N-(pyrrolidine-1-carbonothioyl)-2H-chromene-3-car-
2.2.4. N-(methylcarbamothioyl)-2-oxo-2H-chromene-3-carboxamide
(e1)
boxamide (e6)
Yellow powder, 32% yield; mp. 162–163 ꢁC; IR: 3321, 3051, 2925,
Yellow powder, 52% yield; mp. 146–148 ꢁC; IR: 3363, 3052, 2978, 1704, 1665, 1609, 1523, 1452, 1366, 1163, 761, 610 cmꢀ1
;
1H
1
1698, 1655, 1606, 1562, 1510, 1449, 1243, 1159, 982, 754 cmꢀ1; H
NMR (CDCl3, 300 MHz) d/ppm: 1.91–2.00 (4H, m), 3.46 (2H, t,
J ¼ 6.4 Hz), 3.64 (2H, t, J ¼ 7.0 Hz), 7.27–7.38 (2H, m), 7.53–7.62
NMR (CDCl3, 300 MHz) d/ppm: 3.03 (CH3NH-, 3H, d, J ¼ 4.9 Hz),
7.36–7.43 (2H, m), 7.64–7.76 (2H, m), 8.68 (1H, s, NH), 8.93 (1H, s); (2H, m), 7.97 (1H, s); 13C NMR (CDCl3, 75 MHz) d/ppm:, 24.5,
13C NMR (DMSO-d6, 75 MHz) d/ppm: 27.0, 116.7, 119.1, 119.6,
26.2, 46.4, 47.7, 116.9, 118.5, 123.4, 125.1, 126.4, 128.8, 133.0,
143.3, 154.3, 158.0, 163.4. LC-MS (m/z): 303.3 [Mþ]. Anal. Calcd.
125.7, 130.9, 134.7, 147.9, 148.5, 154.5, 160.9, 162.2. LC-MS (m/z):
300.1 [Mþ]. Anal. Calcd. for C12H10N2O3S; C, 54.95; H, 3.84; N,
for C15H14N2O3S; C, 59.59; H, 4.67; N, 9.27; found: C, 59.55; H,
10.68; found: C, 54.90; H, 3.86; N, 10.67.
4.68; N, 9.29.

1044
B. ZENGIN KURT ET AL.
2.2.10.
2-oxo-N-(piperidine-1-carbonothioyl)-2H-chromene-3-car- 2.2.15. N-((2-morpholinoethyl)carbamothioyl)-2-oxo-2H-chromene-
boxamide (e7)
3-carboxamide (e12)
Yellow powder, 45% yield; mp. 175–176 ꢁC; IR: 3040, 2918, 1710,
Cream powder, 35% yield; mp. 136–138 ꢁC; IR: 3324, 3039, 2916,
1607, 1559, 1438, 1251, 1121, 1041, 757, 610 cmꢀ1 1H NMR
;
1695, 1605, 1542, 1444, 1200, 1113, 765, 644 cmꢀ1 1H NMR
;
(CDCl3, 300 MHz) d/ppm: 1.64 (6H, s, br), 3.34 (2H, s, br), 3.71 (2H,
s, br), 7.29–7.36 (2H, m), 7.52–7.61 (2H, m), 7.87 (1H, s); 13C NMR
(CDCl3, 75 MHz) d/ppm:, 24.5, 25.6, 26.4, 43.2, 48.5, 116.9, 118.6,
125.0, 126.0, 128.6, 132.8, 142.4, 154.1, 158.3, 163.5. LC-MS (m/z):
317.3 [Mþ]. Anal. Calcd. for C16H16N2O3S; C, 60.74; H, 5.10; N, 8.85;
found: C, 60.76; H, 5.12; N, 8.81.
(CDCl3, 300 MHz) d/ppm: 2.52–2.69 (6H, m), 3.56–3.62 (NHCH2-, 2H,
m), 3.74–3.83 (-O(CH2)2, 4H, m), 7.36–7.47 (2H, m), 7.64–7.77 (2H,
m), 8.91 (1H, s), 9.17 (1H, s, NH); 13C NMR (CDCl3, 75 MHz) d/ppm:
36.8, 53.6, 56.9, 67.2, 116.8, 118.7, 118.8, 125.4, 130.0, 134.2, 148.3,
154.6, 161.5, 161.6. LC-MS (m/z): 362.3 [Mþ]. Anal. Calcd. for
C17H19N3O4S; C, 56.50; H, 5.30; N, 11.63; found: C, 56.52; H, 5.32; N,
11.61.
2.2.11.
N-(4-methylpiperazine-1-carbonothioyl)-2-oxo-2H-chro-
2.2.16.
N-((2-(cyclohex-1-en-1-yl)ethyl)carbamothioyl)-2-oxo-2H-
mene-3-carboxamide (e8)
Yellow powder, 35% yield; mp.162–164 ꢁC; IR: 3205, 3040, 2942,
chromene-3-carboxamide (e13)
Yellow powder, 51% yield; mp. 142–143 ꢁC; IR: 3349, 3049, 2926,
1692, 1661, 1607, 1506, 1199, 1122, 792, 551 cmꢀ1 1H NMR
;
1703, 1650, 1610, 1527, 1452, 1243, 1160, 981, 755, 638 cmꢀ1 1H
;
(CDCl3, 300 MHz) d/ppm: 2.36 (3H, s), 2.59 (4H, d, J ¼ 8.2 Hz), 3.69
(2H, s, br), 4.27 (2H, s, br), 7.40–7.46 (2H, m), 7.71–7.77 (2H, m),
8.93 (1H, s), 11.05 (N¼C-SH, 1H, s, SH); 13C NMR (CDCl3, 75 MHz)
d/ppm:, 36.3, 45.9, 51.8, 117.1, 117.5, 118.5, 125.9, 130.3, 135.4,
150.6, 155.0, 157.5, 161.3, 178.1. LC-MS (m/z): 354.2 [Mþ]. Anal.
Calcd. for C16H17N3O3S; C, 57.99; H, 5.17; N, 12.68; found: C, 57.96;
H, 5.19; N, 12.65.
NMR (CDCl3, 300 MHz) d/ppm: 1.55–1.68 (4H, m), 1.98–2.18 (4H, m),
2.25 (-NHCH2CH2-, 2H, t, J ¼ 6.7 Hz), 3.51–3.58 (-NHCH2CH2-, 2H, m),
5.56 (-C ¼ CH, 1H, s), 7.35–7.44 (2H, m), 7.63–7.73 (2H, m), 8.82 (1H,
s, NH), 8.91 (1H, s); 13C NMR (CDCl3, 75 MHz) d/ppm: 22.5, 23.0,
25.4, 28.0, 37.5, 38.2, 116.8, 118.7, 118.8, 124.3, 125.4, 129.9, 134.1,
134.4, 148.3, 154.5, 161.4, 161.5. LC-MS (m/z): 355.3 [Mþ]. Anal.
Calcd. for C19H20N2O3S; C, 64.02; H, 5.66; N, 7.86; found: C, 64.05;
H, 5.61; N, 7.87.
2.2.12. N-(morpholine-4-carbonothioyl)-2-oxo-2H-chromene-3-car-
boxamide (e9)
2.2.17. N-((3,4-dimethoxyphenethyl)carbamothioyl)-2-oxo-2H-chro-
mene-3-carboxamide (e14)
Light yellow powder, 50% yield; mp. 123–125 ꢁC; IR: 3035, 2991,
1714, 1607, 1571, 1428, 1240, 1107, 991, 747, 564 cmꢀ1 1H NMR
;
Yellow powder, 60% yield; mp. 148–149 ꢁC; IR: 3342, 3051, 2934,
(CDCl3, 300 MHz) d/ppm: 3.41 (2H, t, J ¼ 4.9 Hz), 3.72 (2H, t,
J ¼ 4.3 Hz), 3.79 (4H, s), 7.28–7.88 (2H, m), 7.54–7.64 (2H, m), 7.97
(1H, s); 13C NMR (CDCl3, 75 MHz) d/ppm:, 42.7, 47.8, 66.8, 66.9,
117.0, 118.4, 124.9, 125.2, 128.8, 133.3, 144.0, 154.3, 158.2, 163.7.
LC-MS (m/z): 319.3 [Mþ]. Anal. Calcd. for C15H14N2O4S; C, 56.59; H,
4.43; N, 8.80; found: C, 56.55; H, 4.42; N, 8.83.
1
1703, 1655, 1607, 1513, 1451, 1234, 1157, 1029, 747, 641 cmꢀ1; H
NMR (CDCl3, 300 MHz) d/ppm: 2.89 (-NHCH2CH2-, 2H, t, J ¼ 7.3 Hz),
3.67–3.74 (-NHCH2CH2-, 2H, m), 3.86 (-OCH3, 3H, s), 3.90 (-OCH3,
3H, s), 6.79–6.82 (3H, m), 7.36–7.42 (2H, m), 7.64–7.71 (2H, m), 8.89
(1H, s, NH), 8.91 (1H, s); 13C NMR (CDCl3, 75 MHz) d/ppm: 35.4,
41.7, 56.0, 111.4, 112.0, 116.8, 118.5, 118.8, 120.9, 125.5, 130.0,
131.5, 134.2, 147.8, 148.4, 149.1, 154.5, 161.5, 161.6. LC-MS (m/z):
411.3 [Mþ]. Anal. Calcd. for C21H20N2O5S; C, 61.15; H, 4.89; N, 6.79;
found: C, 61.12; H, 4.90; N, 6.76.
2.2.13.
N-((2,3-dihydro-1H-inden-2-yl)carbamothioyl)-2-oxo-2H-
chromene-3- carboxamide (e10)
Yellow powder, 61% yield; mp. 181–183 ꢁC; IR: 3300, 3048, 2953,
1703, 1655, 1606, 1524, 1363, 1201, 797, 742, 632 cmꢀ1 1H NMR
;
2.2.18.
N-((benzo[d][1,3]dioxol-5-ylmethyl)carbamothioyl)-2-oxo-
(CDCl3, 300 MHz) d/ppm: 2.97 (Ar-CH2, 2H, dd, J ¼ 6.1, 16.1 Hz),
3.43 (Ar-CH2, 2H, dd, J ¼ 7.6, 16.1 Hz), 4.86–4.94 (1H, m), 7.17–7.38
(4H, m), 7.41–7.46 (2H, m), 7.63–7.71 (2H, m), 8.92 (1H, s), 9.05 (1H,
d, J¼ 7.0 Hz, NH); 13C NMR (CDCl3, 75 MHz) d/ppm:, 40.1, 51.2,
116.8, 118.6, 118.8, 124.9, 125.0, 125.5, 127.0, 127.1, 130.0, 134.3,
141.0, 148.5, 154.6, 161.5, 161.6. LC-MS (m/z): 385.1 [Mþ]. Anal.
Calcd. for C20H16N2O3S; C, 65.92; H, 4.43; N, 7.69; found: C, 65.90;
H, 4.44; N, 7.68.
2H-chromene-3-carboxamide (e15)
Yellow powder, 66% yield; mp. 188–190 ꢁC; IR: 3352, 3051, 2904,
1706, 1659, 1608, 1498, 1441, 1281, 1237, 1038, 925, 758,
645 cmꢀ1 1H NMR (CDCl3, 300 MHz) d/ppm: 4.56 (-NHCH2-, 2H, d,
;
J ¼ 5.8 Hz), 5.95 (-OCH2O-, 2H, s), 6.76–6.85 (3H, m), 7.36–7.43 (2H,
m), 7.65–7.72 (2H, m), 8.95 (1H, s), 9.12 (1H, s, NH); 13C NMR
(CDCl3, 75 MHz) d/ppm: 43.9, 101.3, 108.5, 108.6, 116.8, 118.5,
118.8, 121.3, 123.4, 125.5, 130.0, 131.9, 134.3, 147.1, 148.1, 148.8,
154.6, 161.6. LC-MS (m/z): 381.2 [Mþ]. Anal. Calcd. for C19H14N2O5S;
C, 59.68; H, 3.69; N, 7.33; found: C, 59.65; H, 3.66; N, 7.35.
2.2.14. 2-oxo-N-((2-(pyrrolidin-1-yl)ethyl)carbamothioyl)-2H-chro-
mene-3-carboxamide (e11)
Cream powder, 42% yield; mp. 131–132 ꢁC; IR: 3326, 3044, 2928,
2.2.19.
boxamide (e16)
N-(morpholinocarbamothioyl)-2-oxo-2H-chromene-3-car-
1698, 1665, 1609, 1542, 1425, 1241, 1147, 995, 760, 642 cmꢀ1 1H
;
Yellow powder, 25% yield; mp. 180–182 ꢁC; IR: 3293, 3233, 3039,
NMR (CDCl3, 300 MHz) d/ppm: 1.80 (4H, s, br), 2.59 (4H, s, br), 2.73
(-NH-CH2CH2N-, 3H, t, J ¼ 6.4 Hz), 3.60–3.64 (-NH-CH2CH2N-, 2H, q,
J ¼ 6.4 Hz), 7.27–7.41 (2H, m), 7.63–7.70 (2H, m), 8.90 (1H, s), 9.03
2987, 1709, 1607, 1528, 1453, 1231, 1107, 865, 761 cmꢀ1 1H NMR
;
(CDCl3, 300 MHz) d/ppm: 2.99–3.08 (-N(CH2)2, 4H, m), 3.80–3.91
(1H, s, NH); 13C NMR (CDCl3, 75 MHz) d/ppm:, 23.8, 39.2, 54.3, 54.8, (-O(CH2)2, 4H, m), 7.38–7.48 (2H, m), 7.67–7.78 (2H, m), 8.96 (1H, s),
116.8, 118.8, 125.4, 129.9, 134.1, 148.3, 154.6, 161.5, 161.7. LC-MS 9.66 (-N¼C-SH, 1H, s, SH); 13C NMR (CDCl3, 75 MHz) d/ppm: 55.0,
(m/z): 385.1 [Mþ]. Anal. Calcd. for C17H19N3O3S; C, 59.11; H, 5.54; N,
56.0, 66.3, 66.5, 116.5, 118.3, 126.0, 130.0, 131.9, 134.5, 149.3,
154.5, 159.3, 161.1, 177.1. LC-MS (m/z): 332.2 [Mþ]. Anal. Calcd. for
12.17; found: C, 59.10; H, 5.53; N, 12.19.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1045
C15H15N3O4S; C, 54.04; H, 4.54; N, 12.60; found: C, 54.07; H, 4.52; N, Na2SO4 or NaClO4 (for maintaining constant the ionic strength;
12.62.
these anions are not inhibitory in the used concentration)26, fol-
lowing the CA-catalysed CO2 hydration reaction for a period of
5–10 s. Saturated CO2 solutions in water at 25 ꢁC were used as
substrate. Stock solutions of inhibitors were prepared at a con-
centration of 10 mM (in DMSO-water 1:1, v/v) and dilutions up
to 1 nM done with the assay buffer mentioned above. At least
seven different inhibitor concentrations have been used for
measuring the inhibition constant. Inhibitor and enzyme solu-
tions were pre-incubated together for 6 h at 4 ꢁC prior to assay,
in order to allow for the formation of the E-I complex. Triplicate
experiments were done for each inhibitor concentration, and the
values reported throughout the paper are the mean of such
results. The inhibition constants were obtained by non-linear
least-squares methods using the Cheng–Prusoff equation, as
reported earlier39 and represent the mean from at least three
different determinations. All CA isozymes used here were recom-
binant proteins obtained as reported earlier by our
2.2.20. N-((4-methylpiperazin-1-yl)carbamothioyl)-2-oxo-2H-chro-
mene-3-carboxamide (e17)
Yellow powder, 38% yield; mp. 204 ꢁC; IR:; IR: 3205, 3040, 2942,
1692, 1661, 1607, 1506, 1199, 1122, 792, 551 cmꢀ1 1H NMR
;
(CDCl3, 300 MHz) d/ppm: 2.37 (-N-CH3, 3H, s), 2.65 (4H, s, br), 3.10
(4H, s, br), 7.43–7.48 (2H, m), 7.74–7.81 (2H, m), 8.94 (1H, s), 11.38
(1H, s, NH), 11.66 (-N¼C-SH, 1H, s, SH); 13C NMR (CDCl3, 75 MHz)
d/ppm: 46.0, 53.9, 54.3, 116.5, 117.3, 118.4, 126.0, 130.5, 136.0,
151.2, 155.1, 160.5, 161.0, 177.0. LC-MS (m/z): 347.3 [Mþ]. Anal.
Calcd. for C16H18N4O3S; C, 55.48; H, 5.24; N, 16.17; found: C, 55.45;
H, 5.22; N, 16.19.
group27,32,40,52
.
2.2.21. 2-oxo-N-(piperidin-1-ylcarbamothioyl)-2H-chromene-3-car-
boxamide (e18)
Orange powder, 50% yield; mp. 206 ꢁC; IR:; IR: 3133, 3047, 2945,
1694, 1607, 1480, 1228, 1190, 1034, 758, 641 cmꢀ1 1H NMR
;
2.4. Molecular modeling
(CDCl3, 300 MHz) d/ppm: 1.50–1.52 (2H, m), 1.74–1.81 (4H, m), 2.99
(4H, s, br), 7.41–7.47 (2H, m), 7.72–7.78 (2H, m), 8.91 (1H, s), 11.32
(1H, s, NH), 11.61 (-N¼C-SH, 1H, s, SH); 13C NMR (CDCl3, 75 MHz)
d/ppm: 23.4, 25.6, 55.1, 55.8, 116.8, 119.0, 119.6, 126.2, 131.5,
135.5, 150.4, 154.8, 159.8, 161.1, 176.4. LC-MS (m/z): 330.3 [Mþ].
Anal. Calcd. for C16H17N3O3S; C, 57.99; H, 5.17; N, 12.68; found: C,
57.97; H, 5.19; N, 12.65.
Molecular modeling approaches such as molecular docking sim-
ulations were performed in terms of examination and compre-
hension of the details in the inhibitory profiles of these
molecules. Binding poses of studied compounds at the binding
pockets of the proteins were determined via molecular docking
processes. The 3D crystal structures of the hCA I, II, VII and IX
were obtained from Protein Data Bank with the corresponding
IDs of 2FW4, 5AML, 3MDZ and 3IAI, respectively. Ligand mole-
cules were two dimensionally sketched in Maestro package of
Schrodinger Small-Molecule Drug Discovery Suite53 and were
prepared via LigPrep54 module of Maestro to establish the con-
formations with the lowest energy in physiological pH 7.4. The
three-dimensional structures of the proteins are also prepared
for docking via Protein Preparation Wizard module of Maestro.
Grid map generation and flexible molecular docking simulations
of ligands to these four proteins were implemented using Glide
module55 and Glide/HTVS (high-throughput virtual screening),
Glide/SP (standard precision), Glide/XP (extra precision), QPLD
(Quantum Mechanics-Polarised Ligand Docking) and IFD
(Induced Fit Docking) protocols of Maestro as well as CCDC
GOLD56 Docking program. As the charge polarisation that
induced by the active site of the protein environment is consid-
ered, quantum mechanics (QM) modeling may give the highest
level of docking accuracy. For these reasons, QPLD is also con-
sidered which uses ab initio charge calculations. Initially, Glide/
SP docking was carried out to generate five poses per docked
compound. These poses were submitted to QM charge calcula-
2.2.22.
2-oxo-N-((2-(piperazin-1-yl)ethyl)carbamothioyl)-2H-chro-
mene-3-carboxamide (e19)
Yellow powder, 32% yield; mp.163–165 ꢁC; IR:; IR: 3205, 3036,
2928, 1695, 1607, 1449, 1225, 1174, 1034, 790, 632 cmꢀ1 1H NMR
;
(CDCl3, 300 MHz) d/ppm: 2.58–2.78 (6H, m), 3.46 (2H, t, J ¼ 4.6 Hz),
3.57–3.62 (2H, q, J ¼ 5.8 Hz), 3.85 (2H, s, br), 7.24–7.42 (2H, m),
7.54–7.71 (2H, m), 7.91 (1H, s), 8.90 (1H, s, NH), 9.18 (1H, s, NH);
13C NMR (CDCl3, 75 MHz) d/ppm: 37.0, 42.4, 47.5, 52.4, 53.0, 56.3,
116.8, 118.5, 125.1, 128.7, 130.0, 134.2, 143.2, 148.4, 154.3, 161.5,
163.5. LC-MS (m/z): 361.3 [Mþ]. Anal. Calcd. for C17H20N4O3S; C,
56.65; H, 5.59; N, 15.54; found: C, 56.63; H, 5.57; N, 15.55.
2.2.23. N-((3-(dimethylamino)propyl)carbamothioyl)-2-oxo-2H-chro-
mene-3-carboxamide (e20)
Yellow powder, 42% yield; mp. 100 ꢁC; IR:; IR: 3341, 3054, 2974,
1704, 1657, 1612, 1451, 1244, 1080, 966, 757, 637 cmꢀ1 1H NMR
;
(CDCl3, 300 MHz) d/ppm: 1.77–1.84 (2H, m), 2.25 (-N(CH3)2, 6H, s),
2.34–2.41 (2H, m), 3.50–3.56 (2H, m), 7.35–7.41 (2H, m), 7.63–7.71
(2H, m), 8.90 (1H, s), 9.08 (1H, s, NH); 13C NMR (CDCl3, 75 MHz)
d/ppm: 27.3, 38.7, 45.6, 57.6, 116.8, 116.8, 118.8X2, 125.4, 129.9,
134.1, 148.3, 154.6, 161.5, 161.6. LC-MS (m/z): 335.3 [Mþ]. Anal.
Calcd. for C16H19N3O3S; C, 57.64; H, 5.74; N, 12.60; found: C, 57.61;
H, 5.76; N, 12.61.
ꢂ
ꢂ
tions, which uses the 6–31 G /LACVP basis set, B3LYP density
functional, and “Ultrafine” SCF accuracy level. In GOLD algo-
rithm, consensus docking protocol was used to generate pro-
tein–ligand complexes with GOLD 5.3.0 software. In this respect,
two docking scoring functions were combined: GoldScore and
ChemScore. In this study, default genetic algorithm parameters
were used and 20 poses were generated for each ligand.
Search efficiency was set to its maximum value (200%) in order
to increase the reliability of the docking results. Flexible amino
2.3. CA inhibition assays
An SX.18 MV-R Applied Photophysics (Oxford, UK) stopped-flow acid side chains/rotatable groups involved in binding pocket
were selected separately for all isoforms according to their pro-
tein–ligand interaction maps available in PDB. Ligand molecules
were also set as flexible during all molecular docking
calculations.
instrument has been used to assay the catalytic/inhibition of
various CA isozymes51. Phenol Red (at
a
concentration of
0.2 mM) has been used as indicator, working at the absorbance
maximum of 557 nm, with 10 mM Hepes (pH 7.4) as buffer, 0.1 M

1046
B. ZENGIN KURT ET AL.
Scheme 1. Synthesis of new thiourea substituted coumaryl-carboxamid derivatives. Reaction conditions: (i) H2O, reflux, 10 h; (ii) SOCl2, 80 ꢁC, 4 h; (iii) KSCN, CH3CN,
70 ꢁC, 30 min.; (iv) RNH2, 70 ꢁC, 4 h.
2,3-dihydro-indenyl ring (e10, Ki¼130.3 nM) led to an increase in
3. Result and discussion
the inhibitory activity against hCA IX; on the other hand, the
3.1. Chemistry
binding of a second alkyl group to -N atom (N,N-diethyl (e3,
Ki ¼ 376.2 nM) and N,N-diisopropyl (e4, Ki ¼ 351.4 nM)) diminished
The syntheses of the target compounds e1–e20 are depicted in
the inhibitory activity against hCA IX. (ii) The expansion of the
pyrrolidine ring of compound e6 (Ki ¼ 297.5 nM against hCA IX)
to a piperidine (compound e7, Ki ¼ 201.8 nM against hCA IX)
increased the inhibitory activity against hCA IX. Additionally,
incorporated N and O atoms into the piperidine ring (R ¼ 4-
methylpiperazine (e8) and R ¼ morpholine (e9), Ki ¼ 128.1 nM
and 136.5 nM, respectively, against hCA IX) caused a greater
increase in the inhibitory activity against hCA IX. (iii) The pres-
ence of an ethyleneamine group as a spacer between the thionyl
moiety and the pyrrolidine ring positively affected the inhibitory
activity against hCA IX (comparing e6 (Ki ¼ 297.5 nM) with e11
(Ki ¼ 107.9 nM)) and the presence of a propyleneamine group
between the thionyl and the N,N-dialkyl moieties did likewise
(e20, Ki ¼ 182.2 nM). On the contrary, the ethyleneamine group
between the thionyl moiety and both the piperazine and mor-
pholine rings decreased the inhibitory activity against hCA IX
(comparing e8 (Ki ¼ 128.1 nM) with e19 (Ki ¼ 249.6 nm) and com-
paring e9 (Ki ¼ 136.5 nm) with e12 (Ki ¼ 223.8 nM)). (iv) Similarly,
the presence of an amine group (-NH-) between the thionyl moi-
ety and the piperidine, piperazine or morpholine ring led to a
major decline the inhibitory activity against hCA IX (comparing
e7 (Ki ¼ 201.8 nM) with e18 (Ki ¼ 387.5 nM), comparing e8
(Ki ¼ 128.1 nM) with e17 (Ki ¼ 258.9 nM) and comparing e9
(Ki ¼ 136.5 nM) with e16 (Ki ¼ 2589.4 nM)). (v) The replacement of
the ethyleneamine group by a methyleneamine between the thi-
onyl moiety and the aromatic ring and the cyclisation of the
dimethoxy group at the phenyl ring to the dioxolane ring did
not cause significant changes in the hCA IX inhibitory activity
(comparing e14 (Ki ¼ 196.4 nM) with e15 (Ki ¼ 184.5 nM)).
Scheme 1. 3-Coumarin carboxylic acid (c) was synthesized from
salicylaldehyde (a) according to literature procedures38 and it was
converted to the acyl chloride by using SOCl2. To obtain thiourea-
substituted coumaryl-carboxamid derivatives (e1–e20), 2-oxo-2H-
chromene-3-carbonyl chloride (d) was reacted with KSCN and
various amines in CH3CN, respectively.
All the new compounds were characterized by 1H NMR, 13C
NMR, IR, MS and elemental analysis. In the IR spectra of the syn-
thesized compounds, it was possible to observe the absorptions
about 3300 cmꢀ1 relating to NH stretch of thiourea groups, about
1650 cmꢀ1 relating to C¼O stretch for thiourea, absorptions in
about 1710 cmꢀ1 from coumarin carbonyl moiety stretch. From
the 1H NMR spectra, the signals for aromatic hydrogens were
observed between 7.17 and 7.77 ppm, the signal of NH proton at
thiourea was detected at about 8.90 ppm and signals observed
about 11.3 ppm for SH proton at the resonance due to thiourea
groups (N¼C–SH). In addition, the signals of aliphatic hydrogen
atoms were determined between 1.00–4.50 ppm. From the 13C
NMR spectra, the signals can be seen about 177 and 163 ppm for
C–SH and carbonyl of thiourea groups, respectively. The signals of
the aliphatic and aromatic carbons were observed at 20–50 ppm
and 110–158 ppm, respectively.
1H NMR, 13C NMR and MS spectra of the synthesized com-
pounds are given in supplementary materials.
3.2. CA inhibition
The inhibition constants (Ki) of the synthesized compounds
e1–e20 against hCA I, hCA II, hCA VII and hCA IX isoforms are
given in Table 1. The hCA I, II and VII isoforms for all compounds
were investigated here in the micromolar range. On the other
hand, the tumour-associated isoform hCA IX was selectively
inhibited by all investigated compounds with inhibition constants
ranging between 107.9 and 2589.4 nM. Compound e11 showed
the strongest inhibition against hCA IX with a Ki of 107.9 nM.
Furthermore, the hCA IX inhibitory activity of e5, e8 and e10 are
close to that of e11 (Ki¼115.1 nM, 128.1 nM and 130.3 nM,
respectively).
The following structure–activity relationship (SAR) observations
can be drawn from data of Table 1: (i) Replacing the methyl
group on the NH of thiourea moiety (e1, Ki¼322.9 nM) by a
propyl (e2, Ki¼286.4 nM), a cyclohexyl (e5, Ki¼115.1 nM) and a
According to X-ray crystallographic studies, coumarins are
mechanism-based inhibitors, which undergo hydrolysis under the
influence of the zinc hydroxide, nucleophilically active species of
the enzyme, with the generation of substituted-2-hydroxycin-
namic acids (Figure 1)26,39–41. It was reported that coumarin/
sulphocoumarin inhibitors and enzyme solutions were pre-
incubated together for ꢃ6 h prior to assay in order to allow for
the formation of the E-I complex or for the eventual active site-
mediated hydrolysis of the inhibitor42. Based on the above con-
sideration, we estimate that the coumarin ring should undergo
ring opening by hydrolysing coumarinic moiety to cinnamic acid
derivative during pre-incubation on enzyme and inhibitor
(Figure 1).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1047
Table 1. Carbonic anhydrase inhibitions of synthesized thiourea substituted coumaryl-carboxamid derivatives.
ꢂ
Ki (nM)
Compound
R
hCA I
hCA II
hCA VII
hCA IX
322.9
e1
>10,000
>10,000
>10,000
e2
e3
>10,000
>10,000
>10,000
>10,000
>10,000
286.4
376.2
>10,000
>10,000
e4
e5
>10,000
>10,000
>10,000
>10,000
351.4
115.1
>10,000
e6
e7
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
297.5
201.8
e8
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
128.1
136.5
130.3
e9
e10
e11
e12
e13
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
107.9
223.8
179.8
(continued)

1048
B. ZENGIN KURT ET AL.
Table 1. Continued
ꢂ
Ki (nM)
Compound
R
hCA I
hCA II
hCA VII
hCA IX
196.4
e14
>10,000
>10,000
>10,000
e15
>10,000
>10,000
>10,000
184.5
e16
e17
e18
e19
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
2589.4
258.9
387.5
249.6
e20
>10,000
>10,000
>10,000
182.2
AAZ
250
12.1
6
25.8
ꢂ
Mean from three different assays, by a stopped flow technique (errors were in the range of 5–10% of the reported
values).
Figure 1. Formation of 2-hydroxy-cinnamic acids A1, B1 and e11-h by the CA-mediated hydrolysis of coumarin A, B and e11.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1049
3.3. Molecular modeling
scores were selected for further analyses. Docking results of com-
pounds (e1–e20) at the binding pockets of hCA isoforms with
Glide/HTVS (high-throughput virtual screening), Glide/SP (standard
precision), Glide/XP (extra precision), IFD (induced fit docking),
QPLD (quantum polarised ligand docking) and GOLD protocols
were compared. Since GOLD docking program gave more success-
ful results GOLD docking results were considered in further ana-
lysis (Tables 2 and 3). Docking scores of studied compounds (with
hydrolyzed and nonhydrolyzed forms) at the hCA I, II, VII and IX
isoforms were compared at the Figures 2 and 3.
The most active compound e11 at the hCA IX showed high
docking scores compared to its predicted binding energies at
hCA I, II and VII isoforms in both hydrolyzed and non-hydrolyzed
forms. In addition, the compounds e2 and e18 in their non-
hydrolyzed forms were found to have high selectivity to hCA IX.
Molecular modeling approaches, such as molecular docking calcu-
lations, are generally used techniques to qualify and quantify the
important information about the ligand–receptor interaction ana-
lysis on atomistic level. There are many studies using these
approaches integrated to human carbonic anhydrases (hCA) in
order to clarify the molecular mechanism of action and bioactive
conformation of proposed compounds at the binding site of the
protein43–49. Since there is no experimental study yet on stable
state (hydrolyzed/nonhydrolyzed) of studied compounds from lac-
tone moiety at the binding pocket of the CAs, both hydrolyzed
and nonhydrolyzed forms were considered at the docking.
Molecular docking results were evaluated for their docking scores
in the binding pocket of the hCA I, II, VII and IX isoforms.
Therefore, the protein–ligand complexes with the top-docking
Table 2. Top-docking scores of compounds e1–e20 (non-hydrolyzed forms) at
the hCAI, hCAII, hCA VII and hCA IX isoforms.
Table 3. Top-docking scores of compounds e1–e20 (hydrolyzed forms) at the
hCAI, hCAII, hCA VII and hCA IX isoforms.
Compounds hCA I (2FW4) hCA II (5AML) hCA VII (3MDZ) hCA IX (3IAI)
Compounds
hCAI (2FW4)
hCAII (5AML)
hCAVII (3MDZ)
hCAIX (3IAI)
e1
e2
e3
e4
e5
e6
e7
e8
ꢀ3.128
ꢀ4.747
ꢀ2.534
ꢀ7.622
ꢀ5.600
ꢀ3.339
ꢀ5.885
ꢀ3.745
ꢀ3.159
ꢀ5.625
ꢀ4.137
ꢀ3.805
ꢀ4.907
ꢀ1.843
ꢀ3.328
ꢀ4.407
ꢀ4.172
ꢀ4.258
ꢀ2.744
ꢀ1.473
ꢀ1.163
ꢀ2.292
ꢀ1.996
ꢀ2.049
ꢀ3.613
ꢀ5.502
ꢀ1.811
ꢀ4.148
ꢀ4.798
ꢀ5.580
ꢀ4.298
ꢀ4.322
ꢀ1.443
ꢀ2.858
ꢀ2.898
ꢀ3.338
ꢀ1.909
ꢀ2.075
ꢀ1.885
0.138
ꢀ5.049
ꢀ4.400
ꢀ5.177
ꢀ6.114
ꢀ4.832
ꢀ5.150
ꢀ5.383
ꢀ5.370
ꢀ6.899
ꢀ6.330
ꢀ4.604
ꢀ6.770
ꢀ5.040
ꢀ5.397
ꢀ4.689
ꢀ5.441
ꢀ4.497
ꢀ4.446
ꢀ4.158
ꢀ4.866
ꢀ4.605
ꢀ8.142
ꢀ5.259
ꢀ4.788
ꢀ4.903
ꢀ5.796
ꢀ4.591
ꢀ6.723
ꢀ6.678
ꢀ5.345
ꢀ8.429
ꢀ4.996
ꢀ5.491
ꢀ5.027
ꢀ6.798
ꢀ6.687
ꢀ4.167
ꢀ8.750
ꢀ5.103
ꢀ4.226
e1
e2
e3
e4
e5
e6
e7
e8
ꢀ4.422
ꢀ4.980
ꢀ4.884
ꢀ5.270
ꢀ5.617
ꢀ4.802
ꢀ4.715
ꢀ4.441
ꢀ5.343
ꢀ4.726
ꢀ4.587
ꢀ5.377
ꢀ5.366
ꢀ5.554
ꢀ4.51
ꢀ5.837
ꢀ6.555
ꢀ4.292
ꢀ7.194
ꢀ5.991
ꢀ6.698
ꢀ6.375
ꢀ3.632
ꢀ6.699
ꢀ7.553
ꢀ5.816
ꢀ5.121
ꢀ6.667
ꢀ5.967
ꢀ5.879
ꢀ6.322
ꢀ5.634
ꢀ6.855
ꢀ6.421
ꢀ5.738
ꢀ5.658
ꢀ5.468
ꢀ6.166
ꢀ4.855
ꢀ6.191
ꢀ6.690
ꢀ5.463
ꢀ5.703
ꢀ6.724
ꢀ7.104
ꢀ5.597
ꢀ7.164
ꢀ5.690
ꢀ6.518
ꢀ6.036
ꢀ6.652
ꢀ6.219
ꢀ3.487
ꢀ7.123
ꢀ6.672
ꢀ7.025
ꢀ6.878
ꢀ7.064
ꢀ1.879
ꢀ8.287
ꢀ7.655
ꢀ6.833
ꢀ4.146
ꢀ7.310
ꢀ8.819
ꢀ9.006
ꢀ8.769
ꢀ9.481
ꢀ9.238
ꢀ9.445
ꢀ4.213
ꢀ4.123
ꢀ2.713
ꢀ9.301
ꢀ9.096
e9
e9
e10
e11
e12
e13
e14
e15
e16
e17
e18
e19
e20
e10
e11
e12
e13
e14
e15
e16
e17
e18
e19
e20
ꢀ4.929
ꢀ4.642
ꢀ5.149
ꢀ5.82
ꢀ4.507
Used protein data bank (PDB) IDs of proteins were also highlighted at the table.
Docking scores are in kcal/mol.
Used protein data bank (PDB) IDs of proteins were also highlighted at the table.
Docking scores are in kcal/mol.
Figure 2. Docking scores (GOLD ChemScore dG) of studied compounds (nonhydrolyzed forms) at the hCA I, II, VII and IX isoforms.

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B. ZENGIN KURT ET AL.
Figure 3. Docking scores (GOLD ChemScore dG) of studied compounds (hydrolyzed forms) at the hCA I, II, VII and IX isoforms.
Figure 4. 2D and 3D ligand interaction diagrams of active compound e11 at the binding cavity of hCA IX. (top) hydrolyzed form; (bottom) nonhydrolyzed form.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1051
Hydrolyzed forms of compounds showed higher docking scores
against hCA IX (i.e. e10–e15, e19 and e20). Figure 4 shows 2D
and 3D ligand interaction diagrams of active compound e11 at
the binding cavity of hCA IX.
sulfamide derivatives incorporating dopaminergic 2-aminote-
tralin scaffolds. Bioorg Med Chem 2016;24:2318–29.
8. Villalba ML, Palestro P, Ceruso M, et al. Sulfamide derivatives
with selective carbonic anhydrase VII inhibitory action.
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drase inhibitor methazolamide prevents amyloid beta-
induced mitochondrial dysfunction and caspase activation
protecting neuronal and glial cells in vitro and in the mouse
brain. Neurobiol Dis 2016;86:29–40.
10. Leppilampi M, Koistinen P, Savolainen ER, et al. The expres-
sion of carbonic anhydrase II in hematological malignancies.
Clin Cancer Res 2002;8:2240–5.
11. Carta F, Osman SM, Vullo D, et al. Dendrimers incorporating
benzenesulfonamide moieties strongly inhibit carbonic anhy-
drase isoforms I–XIV. Org Biomol Chem 2015;13:6453–7.
12. El-Azab AS, Abdel-Aziz AAM, Ayyad RR, et al. Inhibition of
carbonic anhydrase isoforms I, II, IV, VII and XII with carboxy-
lates and sulfonamides incorporating phthalimide/phthalic
anhydride scaffolds. Bioorg Med Chem 2016;24:20–5.
13. Ombouma J, Vullo D, Dumy P, et al. Carbonic Anhydrase
Glycoinhibitors belonging to the Aminoxysulfonamide Series.
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4. Conclusions
A series of 20 novel thiourea-substituted coumaryl-carboxamide
derivatives (e1–20) were synthesized as CA inhibitors and they
were evaluated for the inhibition of hCA I, II, IV and IX isoforms.
All synthesized compounds exhibited selective inhibitory activity
in the high nanomolar range against the tumour-associated iso-
form hCA IX. On the other hand, the hCA I, II and VII isoforms of
were not inhibited by the investigated compounds. Multiscale
molecular modeling approaches and different molecular docking
algorithms were used to investigate inhibitory profiles, binding
poses and predicted binding energies of studied compounds
(both hydrolyzed and non-hydrolyzed forms) at the active sites of
the CA I, II, VII and IX isoforms. The docking studies showed that
hydrolyzed form of e11, which is the most active compound
against hCA IX, interacted with His64, His94, His96 and Gln92 and
non-hydrolyzed form of e11 interacted with His64, His94 and
His119 in the active side of hCA IX.
14. Barresi E, Salerno S, Marini AM, et al. Sulfonamides incorpo-
rating heteropolycyclic scaffolds show potent inhibitory
action against carbonic anhydrase isoforms I, II, IX and XII.
Bioorg Med Chem 2016;24:921–7.
1H and 13C NMR and MS spectra of the synthesized compounds
are given in the Supplementary Materials.
15. Bozdag M, Pinard M, Carta F, et al. A class of 4-sulfamoyl-
phenyl-x-aminoalkyl ethers with effective carbonic anhy-
drase inhibitory action and antiglaucoma effects. J Med
Chem 2014;57:9673–86.
Disclosure statement
No potential conflict of interest was reported by the authors.
16. De Simone G, Supuran CT. Carbonic anhydrase IX: biochem-
ical and crystallographic characterization of a novel antitu-
mor target. Biochim Biophys Acta 2010;1804:404–9.
17. Nishimori I, Vullo D, Minakuchi T, et al. Restoring catalytic
activity to the human carbonic anhydrase (CA) related pro-
teins VIII, X and XI affords isoforms with high catalytic effi-
ciency and susceptibility to anion inhibition. Bioorg Med
Chem Lett 2013;23:256–60.
18. Said HM, Supuran CT, Hageman C, et al. Modulation of car-
bonic anhydrase 9 (CA9) in human brain cancer. Curr Pharm
Des 2010;16:3288–99.
19. Guler OO, De Simone G, Supuran CT. Drug design studies of
the novel antitumor targets carbonic anhydrase IX and XII.
Curr Med Chem 2010;17:1516–26.
20. Akurathi V, Dubois L, Lieuwes NG, et al. Synthesis and bio-
logical evaluation of a 99mTc-labelled sulfonamide conju-
gate for in vivo visualization of carbonic anhydrase IX
expression in tumor hypoxia. Nucl Med Biol 2010;37:557–64.
21. Genis C, Sippel KH, Case N, et al. Design of a carbonic anhy-
drase IX active-site mimic to screen inhibitors for possible
anticancer properties. Biochemistry 2009;48:1322–31.
22. Thiry A, Supuran CT, Masereel B, Dogne JM. Recent develop-
ments of carbonic anhydrase inhibitors as potential anti-
cancer drugs. J Med Chem 2008;51:3051–6.
23. Meijer TW, Bussink J, Zatovicova M, et al. Tumor microenvir-
onmental changes induced by the sulfamate carbonic anhy-
drase IX inhibitor S4 in a laryngeal tumor model. PLoS One
2014;9:e108068.
24. Krall N, Pretto F, Decurtins W, et al. A small molecule drug
conjugate for the treatment of carbonic anhydrase IX
expressing tumors. Angew Chem Int Ed Engl 2014;53:
4231–5.
Funding
This work was supported by the Sakarya Research Fund of the
Sakarya University, 10.13039/501100004473. Project Number:
2016–28-00–003.
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