Coumarin-Thiourea Hybrids Show Potent Carbonic Anhydrase IX and XIII Inhibitory Action

Article abstract of DOI:10.1002/cmdc.202000915

A series of coumarin-thiourea hybrids (4 a–o) has been synthesized, and the compounds have been evaluated against the tumour associated transmembrane isoform, human (h) carbonic anhydrase (CA) hCA IX and the less-explored cytosolic isoform, hCA XIII. All

Full text of DOI:10.1002/cmdc.202000915

Communications
doi.org/10.1002/cmdc.202000915
ChemMedChem
Coumarin-Thiourea Hybrids Show Potent Carbonic
Anhydrase IX and XIII Inhibitory Action
Pavitra S. Thacker,[a] Danaboina Srikanth,[a] Andrea Angeli,[c] Priti Singh,[a]
Krishna Kartheek Chinchilli,[a] Mohammed Arifuddin,*[a, b] and Claudiu T. Supuran*[c]
lungs and metabolizing tissues, biosynthetic reactions, pH
homeostasis, osmotic pressure, ion transport, and neuronal
signalling.[1,2] The CAs have evolved independently into eight
genetically distinct families namely α, β, γ, δ, ζ, η, θ and ι. In
humans, the α-family is present, which consists of 16 isoforms,
among which three are pseudogenes (CAs VIII, X and XI), which
lack the Zn+2 ion at their active site. CA I, II, III, VII, VIII, X, XI and
XIII are cytosolic, CA IV, IX, XII and XIV are transmembrane
isoforms, CAs VA and VB are mitochondrial and CA VI is secreted
in saliva and milk.[1–5]
CA IX is a transmembrane, tumor-associated glycoprotein. It
is activated by a transcription factor, the hypoxia-inducing
factor-1α (HIF-1α), under hypoxic conditions. It is expressed
more in tumor tissues compared to normal tissues and it has
been formally validated as a therapeutic as well as diagnostic
biomarker for solid hypoxic tumors. It functions in pH regulation
of the tumor microenvironment thereby promoting the proc-
esses of tumor cell proliferation, invasion, acquisition of
metastatic phenotype as well as chemoresistance to weakly
basic anticancer drugs. Thus, selective inhibition of CA IX can be
an innovative strategy to target hypoxic tumors, thereby
leading to the treatment of primary tumors and metastasis. Up
until now, various types of molecules belonging to classes such
as peptides, monoclonal antibodies, antibody mimetics and
small molecule inhibitors have been studied for inhibition of
hCA IX.[6–9]
A series of coumarin-thiourea hybrids (4a–o) has been synthe-
sized, and the compounds have been evaluated against the
tumour associated transmembrane isoform, human (h) carbonic
anhydrase (CA) hCA IX and the less-explored cytosolic isoform,
hCA XIII. All compounds exhibited potent inhibition of both
isoforms, with KI values of <100 nM against hCA IX. Compound
4b was the best inhibitor (KI =78.5 nM). All the compounds
inhibited hCA XIII in the low-nanomolar to sub-micromolar
range, with compound 4b again showing the best inhibition
(KI =76.3 nM). With compound 4b as a lead, more-selective
inhibitors of hCA IX and hCA XIII or dual hCA IX/XIII inhibitors
might be developed.
Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are one of the most
widespread and best studied families of enzymes. They belong
to the class of metalloenzymes, as all the eight families known
to date possess a metal ion at their active site. The active site of
this enzyme facilitates the catalysis of a simple but fundamental
reaction across all life forms: the reversible hydration of carbon
dioxide to bicarbonate and protons in
a
two-step
mechanism:[1–4]
(1)
CA XIII, identified in 2004, is a cytosolic CA isoform present
at low concentrations in the submandibular gland, thymus,
small intestine and kidney. Apart from these, it is extensively
located in the reproductive organs. It is present in the testis
during all the stages of sperm development in males and in the
uterine cervix as well as some endometrial glands in females. It
is thought to regulate pH and ion balance, thereby assisting
appropriate fertilization conditions. This isoform possesses a
lower catalytic efficiency as compared to CA II due to lack of a
well-defined cluster of histidine residues in its active site.[10–13]
Coumarin was first isolated in 1820 by Vogel, from the plant
Coumarouna odorata (Dipteryx odorata or tonka bean), and its
derivatives are present as secondary metabolites in many plant
species. Coumarins belong to the lactone class of compounds
and are considered as privileged scaffolds in medicinal
chemistry for possession of a varied pharmacological profile.
Coumarins have been known to possess biological activities like
anticancer, anticoagulant, antimicrobial, anti-inflammatory, anti-
diabetic, anticonvulsant and neuroprotective.[14–16] Coumarins
have been shown to inhibit CAs in a “nonclassical” way, by
occlusion of the active site entrance and undergoing hydrolysis
(2)
where M2+ =ZnII, CdII, FeII or MnII
These enzymes facilitate a large number of pathophysio-
logical processes like transportation of carbon dioxide between
[a] P. S. Thacker, D. Srikanth, P. Singh, K. K. Chinchilli, Dr. M. Arifuddin
Department of Medicinal Chemistry
National Institute of Pharmaceutical Education and Research (NIPER)
Balanagar, Hyderabad, Telangana State (India)
E-mail: arifabib@gmail.com
[b] Dr. M. Arifuddin
Department of Chemistry, Anwarul Uloom College, 11-3-918
New Malleypally, Hyderabad 500001, Telangana State (India)
[c] Dr. A. Angeli, Prof. C. T. Supuran
Università degli Studi di Firenze, Neurofarba Dept.
Sezione di Scienze Farmaceutiche e Nutraceutiche
Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence (Italy)
E-mail: claudiu.supuran@unifi.it
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000915
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to the 2-hydroxy cinnamic acid derivatives.[17,18] Later on, in
particular, they were shown to inhibit selectively and potently,
CAs IX and XII, over other CA isoforms. Over the last decade,
several groups, including ours, have reported coumarins and
their hybrids as selective and potent CA IX inhibitors.[19,20]
Several natural coumarins were reported to be potent inhibitors
of CA XIII.[21]
Thioureas are molecules which possess the fragment NHC
(=S)NH. They have also been known to display diverse bio-
logical profiles like anticancer, anti-inflammatory, antimicrobial
and antiplatelet.[22] Some thioureas have also been reported as
potent CA IX inhibitors.[23]
Hence, in the present study, it was decided to synthesize
coumarin-thiourea hybrids using the principle of molecular
hybridization (Figure 1). The inhibitory potentials of the synthe-
sized molecules were ascertained against the tumor-associated
transmembrane isoform, hCA IX and the cytosolic isoform,
hCA XIII. Acetazolamide (AAZ) was used as the standard drug.
Carbonic anhydrase inhibition
The coumarin-thiourea hybrids were tested for their inhibitory
profile against the transmembrane tumor-associated isoform,
hCA IX and also against the cytosolic isoform, hCA XIII. The
drug, acetazolamide (AAZ), was used as the standard. From the
results shown in Table 1, the following conclusions can be
drawn:
i. The synthesized compounds 4a–o did not show any
inhibition of the cytosolic isoforms hCAs I and II (Ki >
50000 nM).
ii. The transmembrane tumor-associated isoform, hCA IX was
inhibited in a low-to-moderate nanomolar range by com-
pounds 4a–o, with the Ki values ranging from 78.5 to
690.3 nM. The highest inhibition was shown by compound
4b (which has a 4-methoxy substitution on the phenyl ring)
with a Ki value of 78.5 nM. Barring compounds 4d (Ki =
463.5 nM) and 4e (Ki =690.3 nM), all the compounds showed
Ki values of 100 nM or less.
The cytosolic isoform, hCA XIII, was inhibited in a low to
high nanomolar range by the compounds 4a–o, with the Ki
values ranging from 76.3 to 4461 nM. The best inhibition of this
isoform was shown by compound 4b (having a 4-methoxy
substitution on the phenyl ring) with a Ki value of 76.3 nM. The
other compounds with Ki values <100 nM were compounds 4a
(Ki =84.5 nM) and 4h (Ki =94.3 nM), having a 4-methyl and
3,4,5-trimethoxy substitution on the phenyl ring respectively.
All the other compounds showed Ki values >100 nM. It can be
seen that all the three compounds which showed Ki values
<100 nM contained electron-donating substituents on the
phenyl ring.
Results and Discussion
Chemistry
The synthesis of the designed coumarin-thiourea hybrids (4a–
o) was performed according to the general synthetic scheme as
illustrated in Scheme 1. The reduction of 6-nitro coumarin to 6-
amino coumarin (2) and the conversion of 6-amino coumarin to
6-isothiocyanato coumarin (3) was carried out according to the
reported methods.[24,25] Finally, 6-isothiocyanato coumarin was
further reacted with various substituted anilines to afford the
final coumarin-thiourea hybrids 4a–o.
The explanation for the selectivity of the compounds
reported here for inhibiting some CA isoforms (e.g., hCA XII and
Table 1. Inhibition of hCA isoforms I, II, IX and XIII with compounds 4a–o
and AAZ as standard inhibitor.
Compd
R
Ki [nM][a]
hCA I
hCA II
hCA IX
hCA XIII
Figure 1. General structure of the designed coumarin-thiourea hybrids.
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
AAZ
4-CH3
4-OCH3
-H
4-Cl
4-Br
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
250
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
12.1
381.6
78.5
84.0
463.5
690.3
741.0
80.8
87.5
93.2
90.1
96.5
100.0
97.7
91.8
91.3
25.8
84.5
76.3
842.3
802.7
3919
4130
4461
94.3
649.2
6132
606.1
834.5
698.6
521.9
2220
17.0
4-F
3,5-di-OCH3
3,4,5-tri-OCH3
4-phenoxy
4-C(CH3)2
3-Br
3-F
2-Br
4-CF3
2-F
Scheme 1. General synthetic scheme for the synthesis of coumarin-thioureas
°
(4a–o). i) Fe powder, NH4Cl, EtOH/H2O (3:1), 80 C, 2 h; ii) CS2, I2, pyridine,
[a] Mean of 3 different assays, by a stopped flow technique (errors were in
the range of �5–10% of the reported values).
°
0 C, 3 h; iii) substituted aromatic amines, MeCN, RT, 4–5 h.
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°
1 equiv.) in 10 mL of pyridine at 0 C. The contents were stirred for
3 h during which a white solid separated out. The reaction mixture
was distilled until all traces of carbon disulfide and pyridine had
been driven out. Treatment of the residue with dilute HCl rendered
soluble any unaffected amine and pyridine. It was purified using
column chromatography using silica gel 60–120 mesh as the
stationary phase and hexane and ethyl acetate (90:10) as eluent to
afford intermediate 3 as a yellow solid (yield: 80%).
XIII) but not others (hCA I and II) is due to the fact that these
CAIs, presumably in hydrolyzed, substituted-2-hydroxy-cinanmic
acid form, bind in the most variable region of the active site
among the different isoforms, which is the entrance to the
catalytic cleft, as explained by crystallography[17,18] and also
reviewed recently.[1]
Conclusion
Synthesis of compounds 4a–o
To a stirred solution of intermediate 3 (51 mg, 0.25 mmol, 1 equiv.)
in acetonitrile (5ml) was added the appropriate aniline derivative
(0.37 mmol, 1.5 equiv.). The reaction mixture was allowed to stir at
room temperature and the completion of the reaction was
monitored by TLC. Upon completion of the reaction as monitored
by TLC, the reaction solvent was distilled off under vacuum and the
crude residue was subjected to column chromatography using
silica gel 60–120 mesh as the stationary phase and hexane/ethyl-
acetate (70:30) as mobile phase to afford the final coumarin-
thiourea hybrids 4a–o.
In this work, a series of coumarin-thiourea hybrids (4a–o) has
been synthesized and subjected to evaluation against the
transmembrane tumor-associated isoform, human carbonic
anhydrase IX and the cytosolic hCA XIII. The synthesized
compounds exhibited potent inhibition of both hCAs IX and
XIII. For hCA IX, the Ki values ranged from 78.5 to 690.3 nM. The
most potent hCA IX inhibition was shown by compound 4b
possessing a 4-methoxy substitution on the aniline aromatic
ring, with a Ki value of 78.5 nM. For hCA XIII, compound 4b
possessing a 4-methoxy substitution on the aniline aromatic
ring, exhibited the best inhibition, with a Ki value of 76.3 nM.
Hence, compound 4b can be taken as a lead to develop
inhibitors of hCA IX, hCA XIII or both in the cancer treatment.
1-(2-Oxo-2H-chromen-6-yl)-3-(p–tolyl)thiourea (4a): Yield: 75%;
1
°
color: yellow solid; m.p.: 160–162 C; H NMR (500 MHz, [D6]DMSO):
δ=9.7 (s, 1H), 9.77 (s, 1H), 8.08 (d, J=9.2 Hz, 1H), 7.82 (s, 1H), 7.66
(d, J=8.0 Hz, 1H), 7.38 (d, J=7.0 Hz, 3H), 7.22 (d, J=7.0 Hz, 2H),
6.50 (d, J=9.1 Hz, 1H), 2.27 (s, 3H); 13C NMR (125 MHz, [D6]DMSO):
δ=180.65, 160.25, 150.85, 144.59, 137.10, 136.38, 134.44, 129.45,
129.10, 124.54, 123.87, 118.97, 116.76, 116.69, 20.88; HRMS (ESI): m/
z calcd for C17H14N2O2S: 311.0854 [M+H]+; found: 311.0859.
Experimental Section
1-(4-Methoxyphenyl)-3-(2-oxo-2H-chromen-6-yl) thiourea (4b):
1
°
Yield: 58%; color: yellow solid; m.p.: 172–175 C; H NMR (500 MHz,
General
[D6]DMSO): δ=9.71 (s, 1H), 9.70 (s, 1H) 8.07 (d, J=9.6 Hz, 1H), 7.80
(d, J=2.5 Hz, 1H), 7.64 (dd, J=8.9, 2.5 Hz, 1H), 7.38 (d, J=8.9 Hz,
2H), 7.33 (d, J=8.9 Hz, 1H), 6.93 (d, J=9.0 Hz, 2H), 6.50 (d, J=
9.5 Hz, 1H), 3.76 (s, 3H); 13C NMR (125 MHz, [D6]DMSO): δ=180.84,
160.47, 157.21, 150.96, 144.65, 136.41, 132.41, 129.16, 126.64,
123.93, 118.95, 116.76, 116.68, 114.25, 55.73; HRMS (ESI): m/z calcd
for C17H14N2O3S: 326.0725 [M+H]+; found: 365.0381 [M+K]+.
All the chemicals and solvents were procured and used as such
from the suppliers. Wherever necessary, anhydrous solvents were
used. Thin layer chromatography (TLC) analysis was done by
utilizing Merck silica gel 60 F254 aluminum plates. Stuart Digital
Melting Point Apparatus (SMP 30) was used in determining the
1
melting points of the compounds, which are uncorrected. H and
13C NMR spectra were recorded using Bruker Avance 500 MHz and
125 MHz respectively using [D6]DMSO as the solvent. Chemical shift
values are recorded in ppm using TMS as the internal standard.
HRMS were determined by Agilent QTOF mass spectrometer 6540
series instrument and were performed using ESI techniques at
70 eV.
1-(2-Oxo-2H-chromen-6-yl)-3-phenyl)thiourea (4c): Yield: 83%;
1
°
color: yellow solid; m.p.: 150–153 C; H NMR (500 MHz, [D6]DMSO):
δ=9.96 (s, 2H), 8.08 (d, J=9.5 Hz, 1H), 7.82 (s, 1H), 7.65 (d, J=
7.9 Hz, 1H), 7.53 (d, J=8.3 Hz, 2H), 7.48 (d, J=8.6 Hz, 2H), 7.40 (d,
J=8.7 Hz, 2H), 6.51 (d, J=9.5 Hz, 1H); 13C NMR (125 MHz, [D6]
DMSO): δ=180.64, 160.62, 151.08, 144.66, 139.66, 136.37, 129.32,
129.03, 128.95, 125.30, 125.07, 124.47, 124.28, 118.98, 116.79,
116.75; (ESI): m/z calcd for C16H12N2O2S: 297.0698 [M+H]+; found:
Synthesis of Intermediate 2
+
335.0277 [M+K]
.
To a reaction mixture of Fe powder (1.7 g, 31 mmol, 4 equiv.) and
NH4Cl (0.8 g, 15 mmol, 2 equiv.) in EtOH (30 mL) and water (10 mL)
was added 6-nitrocoumarin (1.5 g, 7.8 mmol, 1 equiv.). The resulting
1-(4-Chlorophenyl)-3-(2-oxo-2H-chromen-6-yl) thiourea (4d):
1
°
Yield: 87%; color: yellow solid; m.p.: 155–157 C; H NMR (500 MHz,
°
[D6]DMSO): δ=9.98 (s, 1H), 9.95 (s, 1H), 8.08 (d, J=9.6 Hz, 1H), 7.82
(d, J=2.2 Hz, 1H), 7.65 (dd, J=8.8, 2.3 Hz, 1H), 7.53 (d, J=8.7 Hz,
2H), 7.40 (d, J=8.7 Hz, 3H), 6.50 (d, J=9.6 Hz, 1H); 13C NMR
(125 MHz, [D6]DMSO): δ=180.65, 160.48, 151.14, 144.61, 138.76,
136.07, 129.27, 129.17, 128.84, 128.82, 125.98, 125.85, 124.10,
123.97, 116.85; (ESI): m/z calcd for C16H11ClN2O2S: 331.0308 [M+H]+;
found: 331.0311.
mixture was stirred at 80 C for 2 h. After the reaction was complete,
the reaction mixture was cooled to room temperature and
extracted with ethyl acetate. The organic layer was washed with
brine and dried over Na2SO4. The solid was filtered off, and the
filtrate was concentrated under reduced pressure. The crude
residue was subjected to column chromatography using silica gel
60–120 mesh as the stationary phase and hexane/ethylacetate
(70:30) as eluent to afford intermediate 2 as a yellow solid (yield:
90%).
1-(4-Bromophenyl)-3-(2-oxo-2H-chromen-6-yl)thiourea (4e): Yield:
1
°
95%; color: yellow solid; m.p.: 148–150 C; H NMR (500 MHz, [D6]
DMSO): δ=9.97 (s, 1H), 9.94 (s, 1H), 8.08 (d, J=9.5 Hz, 1H), 7.82 (s,
1H), 7.65 (d, J=7.9 Hz, 1H), 7.53 (d, J=8.3 Hz, 2H), 7.48 (d, J=
8.6 Hz, 2H), 7.40 (d, J=8.7 Hz, 1H), 6.51 (d, J=9.5 Hz, 1H); 13C NMR
(125 MHz, [D6]DMSO): δ=180.57, 160.46, 151.10, 144.61, 139.22,
136.07, 131.76, 129.14, 126.25, 126.11, 124.10, 123.96, 120.72,
Synthesis of Intermediate 3
A solution of iodine (5 mmol 1 equiv.) in 10 mL of carbon disulfide
was added dropwise to a suspension of intermediate 2 (5 mmol
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119.06, 119.05, 116.85; (ESI): m/z calcd for C16H11BrN2O2S: 374.9803
[M+H]+; found: 376.9798.
1-(2-Bromophenyl)-3-(2-oxo-2H-chromen-6-yl)thiourea
(4m):
1
°
Yield: 66%; color: yellow solid; m.p.: 154–157 C; H NMR (500 MHz,
[D6]DMSO): δ=10.06 (s, 1H), 10.00 (s, 1H), 8.09 (d, J=9.6 Hz, 1H),
7.81 (d, J=2.2 Hz, 1H), 7.69 (s, 1H), 7.64 (dd, J=8.8, 2.3 Hz, 1H),
1-(4-Fluorophenyl)-3-(2-oxo-2H-chromen-6-yl)thiourea (4f): Yield:
1
°
67%; color: yellow solid; m.p.: 153–156 C; H NMR (500 MHz, [D6]
7.44–7.33 (m, 3H), 7.20 (d, J=7.8 Hz, 1H), 6.51 (d, J=9.6 Hz, 1H); 13
C
DMSO): δ=9.92 (s, 1H), 9.87 (s, 1H), 8.09 (d, J=8.3 Hz, 1H), 7.82 (s,
1H), 7.66 (s, 1H), 7.48 (s, 2H), 7.40 (d, J=7.8 Hz, 1H), 7.20 (s, 2H), 6.51
(d, J=8.5 Hz, 1H); 13C NMR (125 MHz, [D6]DMSO): δ=180.96, 160.46,
158.80, 151.07, 144.63, 136.19, 136.07, 129.14, 126.89, 126.82,
123.95, 119.04, 116.82, 115.68, 115.51; (ESI): m/z calcd for
C16H11FN2O2S: 315.0604 [M+H]+; found: 337.0412 [M+Na]+.
NMR (125 MHz, [D6]DMSO): δ=180.78, 160.42, 151.23, 144.58,
140.81, 135.83, 130.02, 129.34, 129.16, 127.98, 124.08, 121.28,
120.49, 119.16, 117.00, 116.90; HRMS (ESI): m/z calcd for
C16H11FN2O2S: 374.9803 [M+H]+; found: 376.9776.
1-(2-Oxo-2H-chromen-6-yl)-3-(4-(trifluoromethyl) phenyl)thiourea
(4n): Yield: 76%; color: yellow solid; m.p.: 158–161 C; 1H NMR
°
1-(3,5-Dimethoxyphenyl)-3-(2-oxo-2H-chromen-6-yl)thiourea (4g):
(500 MHz, [D6]DMSO): δ=10.05 (s, 1H), 9.59 (s, 1H), 8.09 (d, J=
9.6 Hz, 1H), 7.84 (d, J=2.4 Hz, 1H), 7.66 (dd, J=8.8, 2.5 Hz, 1H), 7.60
(t, J=7.7 Hz, 1H), 7.40 (d, J=8.8 Hz, 1H), 7.28 (dd, J=9.2, 2.6 Hz,
2H), 7.23–7.18 (m, 1H), 6.51 (d, J=9.6 Hz, 1H); 13C NMR (125 MHz,
[D6]DMSO): δ=180.79, 160.41, 151.23, 144.57, 140.82, 135.85,
130.00, 129.15, 127.94, 124.06, 121.26, 121.23, 120.46, 120.43,
119.16, 116.98, 116.89; HRMS (ESI): m/z calcd for C17H11FN2O2S:
365.0572 [M+H]+; found: 365.0563.
1
°
Yield: 77%; color: yellow solid; m.p.: 164–167 C; H NMR (500 MHz,
[D6]DMSO): δ=10.04 (s, 2H), 8.09 (d, J=9.0 Hz, 1H), 7.83 (s, 1H), 7.66
(s, 1H), 7.39 (d, J=8.2 Hz, 1H), 6.76 (s, 2H), 6.50 (d, J=9.0 Hz, 1H),
6.31 (s, 1H), 3.73 (s, 6H); 13C NMR (125 MHz, [D6]DMSO): δ=180.19,
160.75, 160.46, 151.02, 144.62, 141.31, 136.27, 129.13, 123.88, 118.96,
116.78, 116.70, 102.02, 96.91, 55.68; HRMS (ESI): m/z calcd for
C18H16N2O4S: 357.0909 [M+H]+, found 379.0691 [M+Na]+.
1-(2-Oxo-2H-chromen-6-yl)-3-(3,4,5-trimethoxyphenyl)thiourea
1-(2-Fluorophenyl)-3-(2-oxo-2H-chromen-6-yl) thiourea (4o):
(4h): Yield: 81%; color: yellow solid; m.p.: 161–164 C; 1H NMR
°
1
°
Yield: 61%; color: yellow solid; m.p.: 150–153 C; H NMR (500 MHz,
(500 MHz, [D6]DMSO): δ=9.97 (s, 2H), 8.08 (s, 1H), 7.81 (s, 1H), 7.65
(s, 1H), 7.38 (s, 1H), 6.84 (s, 2H), 6.51 (s, 1H), 3.76 (s, 6H), 3.66 (s, 3H);
13C NMR (125 MHz, [D6]DMSO): δ=180.27, 160.61, 153.05, 151.03,
144.62, 136.31, 135.27, 135.24, 129.27, 124.04, 118.93, 116.78,
116.67, 102.37, 60.52, 56.37; HRMS (ESI): m/z calcd for C19H18N2O5S:
387.1015 [M+H]+, found 409.0786 [M+Na]+.
[D6]DMSO): δ=10.15 (s, 1H), 10.10 (s, 1H), 8.10 (d, J=9.6 Hz, 1H),
7.96 (s, 1H), 7.82 (d, J=2.4 Hz, 1H), 7.78 (d, J=7.9 Hz, 1H), 7.65 (dd,
J=8.8, 2.5 Hz, 1H), 7.58 (t, J=7.9 Hz, 1H), 7.48 (d, J=7.7 Hz, 1H),
7.42 (d, J=8.8 Hz, 1H), 6.52 (d, J=9.6 Hz, 1H);13C NMR (125 MHz,
[D6]DMSO): δ=181.58, 160.44, 157.92, 155.96, 151.13, 144.62,
136.12, 129.15, 128.03, 127.40, 124.69, 124.66, 124.04, 119.03,
116.84, 116.24; HRMS (ESI): m/z calcd for C16H11FN2O2S: 315.0604 [M
+H]+; found: 315.0597.
1-(2-Oxo-2H-chromen-6-yl)-3-(4-phenoxyphenyl) thiourea (4i):
1
°
Yield: 77%; color: yellow solid; m.p.: 170–173 C; H NMR (500 MHz,
[D6]DMSO): Yellow solid9.95 (s, 2H), 8.09 (d, J=8.5 Hz, 1H), 7.82 (s,
1H), 7.65 (d, J=4.3 Hz, 1H), 7.63–7.32 (m, 5H), 7.08 (d, J=64.4 Hz,
5H), 6.51 (d, J=8.6 Hz, 1H); 13C NMR (125 MHz, [D6]DMSO): Yellow
solid180.71, 160.45, 157.40, 153.91, 150.99, 144.64, 136.29, 135.24,
130.50, 129.11, 126.44, 123.87, 123.79, 119.31, 118.78, 116.80,
116.75; HRMS (ESI): m/z calcd for C22H16N2O3S: 389.0960 [M+H]+;
found: 389.0908.
CA inhibition
An SX.18V-R Applied Photophysics (Oxford, UK) stopped flow
instrument was used to assay the catalytic/inhibition of various CA
isozymes.[26] Phenol Red (at a concentration of 0.2 mM) has been
used as an indicator, working at an absorbance maximum of
557 nm, with 10 mM HEPES (pH 7.4) as a buffer, 0.1M Na2SO4 or
NaClO4 (for maintaining constant the ionic strength; these anions
are not inhibitory in the used concentration), following the CA-
catalyzed CO2 hydration reaction for a period of 5–10 s. Saturated
1-(4-Isopropylphenyl)-3-(2-oxo-2H-chromen-6-yl)thiourea
(4j):
1
°
Yield: 95%; color: yellow solid; m.p.: 178–181 C; H NMR (500 MHz,
[D6]DMSO): δ=9.86 (s, 2H), 8.08 (d, J=9.2 Hz, 1H), 7.82 (s, 1H), 7.66
(d, J=8.0 Hz, 1H), 7.38 (d, J=7.0 Hz, 3H), 7.22 (d, J=7.0 Hz, 2H),
6.50 (d, J=9.1 Hz, 1H), 2.88 (s, 1H), 1.20 (s, 6H); 13C NMR (125 MHz,
[D6]DMSO): δ=180.64, 160.62, 151.08, 144.66, 139.66, 136.37,
129.32, 129.03, 128.95, 125.30, 125.07, 124.47, 124.28, 118.98,
116.79, 116.75; HRMS (ESI): m/z calcd for C19H18N2O2S: 339.1167 [M
+H]+; found: 339.1110.
°
CO2 solutions in water at 25 C were used as substrate. Stock
solutions of inhibitors were prepared at a concentration of 10 mM
(in DMSO/water 1:1, v/v) and dilutions up to 0.01 nM done with the
assay buffer mentioned above. At least 7 different inhibitor
concentrations have been used for measuring the inhibition
constant. Inhibitor and enzyme solutions were pre-incubated
1-(3-Bromophenyl)-3-(2-oxo-2H-chromen-6-yl)thiourea (4k): Yield:
°
together for 6 h at 4 C prior to assay, in order to allow for the
1
°
83.52%; color: yellow solid; m.p.: 150–153 C; H NMR (500 MHz, [D6]
formation of the E-I complex. Triplicate experiments were done for
each inhibitor concentration, and the values are reported through-
out the paper is the mean of such results. The inhibition constants
were obtained by nonlinear least squares methods using the
Cheng-Prusoff equation, as reported earlier, and represent the
mean from at least three different determinations. All CA isozymes
used here were recombinant proteins obtained as reported earlier
by our groupand their concentration in the assay system was of 6–
12 nM.[27,28]
DMSO): δ=10.07 (s, 1H), 10.04(s, 1H), 8.10 (d, J=10.3 Hz, 1H), 7.83
(s, 2H), 7.66 (s, 1H), 7.39 (dd, J=55.4, 19.1 Hz, 3H), 6.52 (s, 1H), 5.77
(d, J=10.1 Hz, 1H);13C NMR (125 MHz, [D6]DMSO): δ=180.61,
160.43, 151.17, 144.60, 141.55, 135.98, 130.82, 129.15, 127.55,
126.51, 124.02, 123.02, 121.41, 119.10, 116.90, 116.87; HRMS (ESI):
m/z calcd for C16H11BrN2O2S: 374.9803 [M+H]+; found: 414.9358 [M
+K]+.
1-(3-Fluorophenyl)-3-(2-oxo-2H-chromen-6-yl)thiourea (4l): Yield:
1
°
81%; color: yellow solid; m.p.: 150–153 C; H NMR (500 MHz, [D6]
DMSO): δ=10.10 (s, 1H), 10.09 (s, 1H), 8.09 (d, J=9.6 Hz, 1H), 7.83
(s, 1H), 7.66 (dd, J=8.7, 1.9 Hz, 1H), 7.55 (d, J=11.2 Hz, 1H), 7.45–
7.34 (m, 2H), 7.28 (d, J=7.9 Hz, 1H), 6.98 (d, J=6.9 Hz, 1H), 6.51 (d,
J=9.5 Hz, 1H); 13C NMR (125 MHz, [D6]DMSO): δ=180.78, 160.42,
151.23, 144.58, 140.81, 135.83, 130.02, 129.34, 129.16, 127.98,
124.08, 121.28, 120.49, 119.16, 117.00, 116.90; HRMS (ESI): m/z calcd
for C16H11FN2O2S: 315.0604 [M+H]+; found: 315.0614.
Acknowledgements
P.S.T., D.S., P.S. and K.K.C. would like to thank the Department of
Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of
India, New Delhi for the award of a NIPER Fellowship.
ChemMedChem 2021, 16, 1252–1256
www.chemmedchem.org
1255
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Communications
doi.org/10.1002/cmdc.202000915
ChemMedChem
[18] A. Maresca, C. Temperini, L. Pochet, B. Masereel, A. Scozzafava, C. T.
Conflict of Interest
Supuran, J. Med. Chem. 2010, 53, 335–44.
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The authors declare no conflict of interest.
Keywords: carbonic anhydrases · coumarin · hCA · thiourea
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Manuscript received: November 24, 2020
Accepted manuscript online: December 21, 2020
Version of record online: February 9, 2021
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ChemMedChem 2021, 16, 1252–1256
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