7-Acylamino-3H-1,2-benzoxathiepine 2,2-dioxides as new isoform-selective carbonic anhydrase IX and XII inhibitors

Article abstract of DOI:10.1080/14756366.2020.1722658

A series of 3H-1,2-benzoxathiepine 2,2-dioxides incorporating 7-acylamino moieties were obtained by an original procedure starting from 5-nitrosalicylaldehyde, which was treated with propenylsulfonyl chloride followed by Wittig reaction of the bis-olefin intermediate. The new derivatives, belonging to the homosulfocoumarin chemotype, were assayed as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Four pharmacologically relevant human (h) isoforms were investigated, the cytosolic hCA I and II and the transmembrane, tumour-associated hCA IX and XII. No relevant inhibition of hCA I and II was observed, whereas some of the new derivatives were effective, low nanomolar hCA IX/XII inhibitors, making them of interest for investigations in situations in which the activity of these isoforms is overexpressed, such as hypoxic tumours, arthritis or cerebral ischaemia.

Full text of DOI:10.1080/14756366.2020.1722658

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
7-Acylamino-3H-1,2-benzoxathiepine 2,2-dioxides
as new isoform-selective carbonic anhydrase IX
and XII inhibitors
Aleksandrs Pustenko, Alessio Nocentini, Anastasija Balašova, Mikhail
Krasavin, Raivis Žalubovskis & Claudiu T. Supuran
To cite this article: Aleksandrs Pustenko, Alessio Nocentini, Anastasija Balašova, Mikhail
Krasavin, Raivis Žalubovskis & Claudiu T. Supuran (2020) 7-Acylamino-3H-1,2-benzoxathiepine
2,2-dioxides as new isoform-selective carbonic anhydrase IX and XII inhibitors, Journal of Enzyme
Inhibition and Medicinal Chemistry, 35:1, 650-656, DOI: 10.1080/14756366.2020.1722658
To link to this article: https://doi.org/10.1080/14756366.2020.1722658
©
2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 21 Feb 2020.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
020, VOL. 35, NO. 1, 650–656
2
https://doi.org/10.1080/14756366.2020.1722658
RESEARCH PAPER
7
-Acylamino-3H-1,2-benzoxathiepine 2,2-dioxides as new isoform-selective
carbonic anhydrase IX and XII inhibitors
a,b
c
a
d
a,b
ꢀ
Aleksandrs Pustenko , Alessio Nocentini , Anastasija Bala ꢀs ova , Mikhail Krasavin , Raivis Zalubovskis and
Claudiu T. Supuran
c
a
b
Latvian Institute of Organic Synthesis, Riga, Latvia; Institute of Technology of Organic Chemistry, Faculty of Materials Science and Applied
c
Chemistry, Riga Technical University, Riga, Latvia; Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universit ꢁa degli
Studi di Firenze, Florence, Italy; Department of Chemistry, Saint Petersburg State University, Saint Petersburg, Russian Federation
d
ABSTRACT
ARTICLE HISTORY
A series of 3H-1,2-benzoxathiepine 2,2-dioxides incorporating 7-acylamino moieties were obtained by an
original procedure starting from 5-nitrosalicylaldehyde, which was treated with propenylsulfonyl chloride
followed by Wittig reaction of the bis-olefin intermediate. The new derivatives, belonging to the homosul-
focoumarin chemotype, were assayed as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC
Received 26 December 2019
Revised 21 January 2020
Accepted 23 January 2020
KEYWORDS
4
.2.1.1). Four pharmacologically relevant human (h) isoforms were investigated, the cytosolic hCA I and II
Carbonic anhydrase;
transmembrane isoforms;
sulfocoumarin; homosulfo-
coumarin; isoform-
selective inhibitor
and the transmembrane, tumour-associated hCA IX and XII. No relevant inhibition of hCA I and II was
observed, whereas some of the new derivatives were effective, low nanomolar hCA IX/XII inhibitors, mak-
ing them of interest for investigations in situations in which the activity of these isoforms is overex-
pressed, such as hypoxic tumours, arthritis or cerebral ischaemia.
1
. Introduction
least 12 catalytically active and three acatalytic isoforms are pre-
1
1,12
sent
. However, the new generation CAIs to which coumarins
Sulfocoumarins (1,2-benzoxathiine 2,2-dioxides) and homosulfo-
1
–5
and sulfocoumarins belong, show significant isoform-selective
inhibition profiles, as demonstrated in a considerable number of
coumarins (3H-1,2-benzoxathiepine 2,2-dioxides)
most investigated new classes of carbonic anhydrase (CA, EC
.2.1.1) inhibitors, which have been designed considering the
are among the
1–8
studies . This is principally due to the fact that these com-
pounds possess a distinct inhibition mechanism compared to the
sulphonamides, which coordinate to the zinc ion from the CA
4
6–8
structurally similar coumarins as lead molecules. Indeed, Cas are
widely spread enzymes in organisms of all types, from simple to
complex ones
1
1,12
9–15
active site as anions
. In fact, coumarins and sulfocoumarins
, and are involved in crucial physiological proc-
act as prodrug inhibitors, undergoing an active site mediated
hydrolysis, which leads to the formation of 2-hydroxy-cinnamic
acids in the case of the coumarins, and ethane-sulphonates in the
case of the sulfocoumarins, which subsequently bind in different
active site regions, different of those where the classical sulphona-
esses, among which carbon fixation in diatoms and other marine
organisms in which several genetic families of such metalloen-
9
zymes were reported . In protozoans, Cas are involved in biosyn-
9
thetic reactions whereas in bacteria, where at least three genetic
families were described (a-, b-, and c-Cas) these enzymes play cru-
1–8
mide CAIs bind . As shown by X-ray crystallography, the hydro-
lysed coumarins occlude the entrance of the CA active site
cial roles related both to metabolism but also virulence and sur-
10
vival in various niches . In vertebrates, including humans, a high
6
cavity , whereas the sulfocoumarins bind deeper within the active
number of different CA isoforms belonging to the a-CA class were
1
1,12
site, but still do not coordinate to the metal ion. Instead, the
formed sulphonates anchor to the zinc-coordinated water mol-
ecule, as shown again by means of X-ray crystallographic techni-
described
2
, which by hydrating CO to a weak base (bicarbon-
ate) and a strong acid (hydronium ions), are involved in a multi-
tude of processes, starting with pH regulation and ending with
2
1
3,14
ques . As these regions of the CA active site are the most variable
metabolism
. As thus, Cas are drug targets for decades, with
their inhibitors having pharmacological applications in a multitude ones, a straightforward explanation of the isoform selectivity of
1
1–16
of fields
. The primary sulphonamides were discovered as CA these new generation CAIs was furnished by using a combination
inhibitors (CAIs) in the ‘40 s, and most of the drugs that were of crystallographic and kinetic studies, which also allowed the
launched in the next decades as diuretics, antiepileptics, or anti- development of compounds showing a higher degree of selectiv-
1
5,16
glaucoma agents belonged to this class of compounds or to their ity
. This allowed for the development of inhibitors useful for
11
isosteres such as the sulfamates and sulfamides . An important new pharmacological applications such as antitumor/antimeta-
drawback of such first generation CA inhibitors (CAIs) was their static compounds , CAIs useful for the management of arthritis ,
lack of isoform selectivity, considering the fact that in humans at neuropathic pain , and cerebral ischaemia .
1
3
17
1
8
19
ꢀ
CONTACT Raivis Zalubovskis
claudiu.supuran@unifi.it
Florence, Italy
raivis@osi.lv
Latvian Institute of Organic Synthesis, 21 Aizkraukles Str, Riga, LV-1006, Latvia; Claudiu T. Supuran
Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universit ꢁa degli Studi di Firenze, Sesto Fiorentino,
ß 2020 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,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
651
Considering our interest in designing non-sulphonamide CAIs Et
with various potential applications, we report here a new series of Mp 174–175 C.
3
N
(110 mL; 0.78 mmol) as white solid (162 mg; 72%).
ꢀ
ꢂ1
homosulfocoumarins and their inhibitory profiles against the
major human (h) CA isoforms, hCA I, II, IX, and XII, involved in 1363 (S ¼ O), 1163 (S ¼ O);
IR (film, cm ) ꢀmax¼ 3289 (N–H), 1652 (C ¼ O), 1370 (S ¼ O),
1
many pathologies, including cancer.
H NMR (400 MHz, DMSO-d
6
) d ¼ 4.43 (dd, 2H, J ¼ 6.0, 0.9 Hz),
5
7
7
.99–6.06 (m, 1H), 6.93 (d, 1H, J ¼ 11.2 Hz), 7.35 (d, 1H, J ¼ 8.8 Hz),
.52–7.58 (m, 2H), 7.59–7.64 (m, 1H), 7.82 (dd, 1H, J ¼ 8.8, 2.5 Hz),
.91 (d, 1H, J ¼ 2.5 Hz), 7.94–7.99 (m, 2H), 10.46 (s, 1H) ppm.
2
. Experimental part
1
3
C NMR (100 MHz, DMSO-d
6
) d ¼ 51.1, 120.9, 122.0, 122.1,
2
.1. Chemistry
1
1
22.6, 127.7, 128.3, 128.5, 131.4, 131.8, 134.6, 137.9,
42.7, 165.7 ppm
Reagents, starting materials/intermediates 1–7 and solvents were
obtained from commercial sources (Sigma-Aldrich, St. Louis, MO)
þ
HRMS (ESI) [M þ H] : m/z calcd for (C H NO S) 316.0644.
1
6
14
4
2 2
and used as received. Anhydrous CH Cl and toluene were
Found 316.0654.
obtained by passing commercially available solvents through acti-
vated alumina columns. Thin-layer chromatography was per-
formed on silica gel, spots were visualised with UV light (254 and
N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)-4-methy benzamide (10).
3
65 nm). Melting points were determined on an OptiMelt auto-
H
N
Compound 10 was prepared
according to the general procedure
from amino derivative 7 (150 mg;
mated melting point system. IR spectra were recorded on
Shimadzu FTIR IR Prestige-21 spectrometer. NMR spectra were
recorded on Bruker Avance Neo (400 MHz) spectrometer with
chemical shifts values (d) in ppm relative to TMS using the
residual DMSO-d signal ( H 2.50; C 39.52) or CDCl3 signal ( H
7
O
SO2
O
0
.71 mmol), 4-methylbenzoyl chlor-
ide (103 mL; 0.78 mmol) and Et
3
N (110 mL; 0.78 mmol) as white crys-
tals (170 mg; 73%). Mp 197–198 C.
1
13
1
ꢀ
6
1
3
ꢂ1
.26;
C 77.16) as an internal standard. High-resolution mass
IR (film, cm ) ꢀmax ¼ 3324 (N–H), 1646 (C ¼ O), 1378 (S ¼ O),
spectra (HRMS) were recorded on a mass spectrometer with a Q- 1363 (S ¼ O), 1177 (S ¼ O), 1169 (S ¼ O);
1
TOF micro mass analyser using the ESI technique.
H NMR (400 MHz, DMSO-d ) d ¼ 2.39 (s, 3H), 4.41–4.45 (m, 2H),
6
5
(
.99–6.06 (m, 1H), 6.92 (d, 1H, J ¼ 11.2 Hz), 7.32–7.37 (m, 3H), 7.82
dd, 1H, J ¼ 8.9, 2.6 Hz), 7.86–7.92 (m, 3H), 10.37 (s, 1H) ppm
General procedure for synthesis of acyl compound 8–17
To a solution of amino derivative 7 (1.0 eq.) in dry CH
per mmol of compound 7) at 0 C appropriate acyl chloride (1.1
eq.) and Net
stirred at room temperature under an argon atmosphere for 2 h.
Water was added (20 ml per mmol of compound 7). Layers were
separated, water layer was washed with EtOAc (2 ꢁ 40 ml).
Combined organic layers were washed with brine, dried over anh.
2 4
Na SO , filtered, evaporated. The crude solids were recrystallised
form EtOAc/petrol ether mixture to afford product.
13
C NMR (100 MHz, DMSO-d
22.6, 127.7, 128.3, 129.0, 131.4, 131.6, 138.0, 141.9,
42.6, 165.5 ppm
6
) d ¼ 21.0, 51.1, 120.8, 121.9, 122.1,
Cl
2 2
(20 ml
1
1
ꢀ
(1.1 eq.) were added. The resulting mixture was
þ
3
HRMS (ESI) [M þ H] : m/z calcd for (C H NO S) 330.0800.
1
7
16
4
Found 330.0815.
N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)4-bromobenzamide (11).
Br
Compound 11 was prepared
H
N
according to the general proced-
ure from amino derivative
7
O
SO
O
2 (150 mg; 0.71 mmol), 4-bromoben-
N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)acetamide (8).
zoyl chloride (171 mg; 0.78 mmol) and Et N (110 mL; 0.78 mmol) as
3
H
Compound 8a was prepared according to
the general procedure from amino deriva-
tive 7 (150 mg; 0.71 mmol), acetyl chloride
ꢀ
white solid (166 mg; 59%). Mp 185–186 C.
N
ꢂ1
IR (film, cm ) ꢀmax¼ 3260 (N–H), 1653 (C ¼ O), 1375 (S ¼ O),
O
1
363 (S ¼ O), 1167 (S ¼ O);
SO2
O
1
(
56 mL; 0.78 mmol) and Et N (110 mL;
H NMR (400 MHz, DMSO-d ) d ¼ 4.42–4.46 (m, 2H), 5.99–6.06
3
ꢀ
6
0
1
5
.78 mmol) as white solid (127 mg; 70%). Mp 164–165 C.
(m, 1H), 6.92 (d, 1H, J ¼ 11.3 Hz), 7.35 (d, 1H, J ¼ 8.8 Hz), 7.74–7.83
ꢂ1
IR (film, cm ) ꢀmax¼ 3276 (N–H), 1670 (C ¼ O), 1370 (S ¼ O), (m, 3H), 7.88–7.94 (m, 3H), 10.52 (s, 1H) ppm
1
3
361 (S ¼ O), 1166 (S ¼ O), 1162 (S ¼ O);
C NMR (100 MHz, DMSO-d ) d ¼ 51.2, 120.9, 122.0, 122.2,
6
1
H NMR (400 MHz, DMSO-d
6
) d ¼ 2.06 (s, 3H), 4.37–4.41 (m, 2H), 122.7, 125.6, 128.3, 129.8, 131.4, 131.5, 133.6, 137.7,
.96–5.6.04 (m, 1H), 6.89 (d, 1H, J ¼ 11.3 Hz), 7.28 (d, 1H, 142.8, 164.7 ppm
þ
J ¼ 8.9 Hz), 7.58 (dd, 1H, J ¼ 8.9, 2.5 Hz), 7.69 (d, 1H, J ¼ 2.5 Hz),
HRMS (ESI) [M þ H] : m/z calcd for (C H BrNO S) 393.9749.
1
6
13
4
1
0.16 (s, 1H) ppm.
Found 393.9736.
1
3
C NMR (100 MHz, DMSO-d
6
) d ¼ 24.0, 51.0, 120.6, 120.8, 122.7,
1
28.4, 131.5, 138.0, 142.2, 168.6 ppm.
N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)-2-iodobenzamide (12).
þ
HRMS (ESI) [M þ H] : m/z calcd for (C H NO S) 254.0487.
11
12
4
Found 254.0498.
Compound 12 was prepared
according to the general procedure
from amino derivative 7 (150 mg;
0.71 mmol), 2-iodobenzoyl chloride
N (110 mL; 0.78 mmol) as white solid
3
H
N
N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)benzamide (9).
I
O
SO2
O
H
Compound 9 was prepared according (208 mg; 0.78 mmol) and Et
N
ꢀ
to the general procedure from amino (276 mg; 88%). Mp 188–189 C.
ꢂ1
derivative 7 (150 mg; 0.71 mmol), ben-
IR (film, cm ) ꢀmax¼ 3240 (N–H), 1641 (C ¼ O), 1374 (S ¼ O),
O
SO
O
2 zoyl chloride (90 mL; 0.78 mmol) and 1362 (S ¼ O), 1156 (S ¼ O);

6
52
A. PUSTENKO ET AL.
1
1
H NMR (400 MHz, DMSO-d
6
) d ¼ 4.41–4.45 (m, 2H), 6.00–6.08
H NMR (400 MHz, DMSO-d
6
) d ¼ 4.42–4.45 (m, 2H), 6.00–6.08
(
m, 1H), 6.94 (d, 1H, J ¼ 11.2 Hz), 7.22–7.28 (m, 1H), 7.36 (d, 1H, (m, 1H), 6.94 (d, 1H, J ¼ 11.2 Hz), 7.36 (d, 1H, J ¼ 8.8 Hz), 7.67–7.76
J ¼ 8.8 Hz), 7.47–7.55 (m, 2H), 7.72 (dd, 1H, J ¼ 8.8, 2.5 Hz), 7.87 (d, (m, 3H), 7.78–7.89 (m, 3H), 10.81 (s, 1H) ppm
1
3
1
H, J ¼ 2.5 Hz), 7.9–7.97 (m, 1H), 10.67 (s, 1H) ppm
C NMR (100 MHz, DMSO-d ) d ¼ 51.0, 121.1, 121.3, 122.9,
6
1
3
C NMR (100 MHz, DMSO-d
22.9, 128.1, 128.2, 128.5, 131.2, 131.5, 137.7, 139.1, 142.7, 128.5, 128.6, 130.3, 131.4, 132.7, 135.8 (q, J ¼ 2.3 Hz), 137.6,
42.8, 167.7 ppm
142.8, 165.8 ppm
HRMS (ESI) [M þ H] : m/z calcd for (C H NO F3S) 384.0517
6
) d ¼ 51.0, 93.6, 121.0, 121.2, 121.3, 123.8 (q, J ¼ 274 Hz), 125.8 (q, J ¼ 31.2 Hz), 126.4 (q, J ¼ 4.6 Hz),
1
1
þ
þ
HRMS (ESI) [M þ H] : m/z calcd for (C H INO S) 441.9610
16
13
4
17 13
4
Found 441.9609.
Found 384.0519.
N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)thiophene-2-carboxa-
N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)-2-bromobenzamide (13).
mide (16).
H
N
Compound 13 was prepared accord-
Compound 16 was prepared accord-
ing to the general procedure from
H
ing to the general procedure from
amino derivative (150 mg;
.71 mmol), 2-thiophenecarbonyl
N
amino
derivative
7
(150 mg;
S
Br
O
7
SO2
O
0.71 mmol), 2-bromobenzoyl chloride
O
0
SO2
O
(
(
102 mL; 0.78 mmol) and Et
230 mg; 82%). Mp 177–178 C.
3
ꢀ
N (110 mL; 0.78 mmol) as white solid
chloride (84 mL; 0.78 mmol) and Et N
3
ꢀ
(
110 mL; 0.78 mmol) as white solid (185 mg; 81%). Mp 162–163 C.
ꢂ1
IR (film, cm ) ꢀmax¼ 3288 (N–H), 1653 (C ¼ O), 1371 (S ¼ O),
ꢂ1
IR (film, cm ) ꢀmax¼ 3357 (N–H), 1648 (C ¼ O), 1372 (S ¼ O),
1
176 (S ¼ O), 1156 (S ¼ O);
1
356 (S ¼ O), 1178 (S ¼ O), 1165 (S ¼ O);
1
H NMR (400 MHz, DMSO-d
6
) d ¼ 4.43 (dd, 2H, J ¼ 6.0, 0.9 Hz),
1
H NMR (400 MHz, DMSO-d ) d ¼ 4.44 (dd, 2H, J ¼ 6.0, 1.1 Hz),
6
6
7
7
.00–6.07 (m, 1H), 6.94 (d, 1H, J ¼ 11.2 Hz), 7.36 (d, 1H, J ¼ 8.9 Hz),
.41–7.47 (m, 1H), 7.51 (dt, 1H, J ¼ 7.4, 1.1 Hz), 7.55–7.59 (m, 1H),
.69–7.76 (m, 2H), 7.87 (d, 1H, J ¼ 2.6 Hz), 10.73 (s, 1H) ppm
5
(
.99–6.06 (m, 1H), 6.92 (d, 1H, J ¼ 11.2 Hz), 7.23–7.26 (m, 1H), 7.35
d, 1H, J ¼ 8.8 Hz), 7.78 (dd, 1H, J ¼ 8.8, 2.6 Hz), 7.84 (d, 1H,
J ¼ 2.6 Hz), 7.88 (dd, 1H, J ¼ 5.0, 1.1 Hz), 8.04 (dd, 1H, J ¼ 3.8,
1
3
C NMR (100 MHz, DMSO-d ) d ¼ 51.0, 118.9, 121.1, 121.2,
6
1
.1 Hz), 10.43 (s, 1H) ppm
1
1
22.9, 127.8, 128.6, 128.9, 131.4, 131.5, 132.8, 137.6, 138.8,
42.8, 166.0 ppm
13
C NMR (100 MHz, DMSO-d ) d ¼ 51.2, 120.9, 121.9, 122.1,
6
1
1
22.7, 128.2, 128.4, 129.5, 131.3, 132.3, 137.5, 139.5,
þ
HRMS (ESI) [M þ H] : m/z calcd for (C H BrNO S) 393.9749
16
13
4
42.7, 160.0 ppm
Found 393.9766.
þ
HRMS (ESI) [M þ H] : m/z calcd for (C14
4 2
H12NO S ) 322.0208
Found 322.0221.
N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)-2-fluorobenzamide (14).
F
Compound 14 was prepared accord- N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)furan-2-carboxamide (17).
H
N
ing to the general procedure from
amino derivative (150 mg;
SO2 0.71 mmol), 2-fluorobenzoyl chloride
Compound 17 was prepared according
to the general procedure from amino
derivative 7 (150 mg; 0.71 mmol), 2-
furoyl chloride (84 mL; 0.78 mmol) and
H
N
7
O
O
O
(
(
93 mL; 0.78 mmol) and Et
188 mg; 79%). Mp 173–174 C.
3
N (110 mL; 0.78 mmol) as white solid
O
SO2
ꢀ
O
Et
3
N (110 mL; 0.78 mmol) as white solid
ꢂ1
IR (film, cm ) ꢀmax¼ 1671 (C ¼ O), 1371 (S ¼ O), 1365 (S ¼ O),
ꢀ
(
185 mg; 81%). Mp 162–163 C.
ꢂ1
1
164 (S ¼ O) 1156 (S ¼ O);
1
IR (film, cm ) ꢀmax¼ 3299 (N–H), 1663 (C ¼ O), 1367 (S ¼ O),
H NMR (400 MHz, DMSO-d ) d ¼ 4.41–4.45 (m, 2H), 6.00–6.07
6
1363 (S ¼ O), 1165 (S ¼ O), 1158 (S ¼ O);
1
(
1
m, 1H), 6.93 (d, 1H, J ¼ 11.2 Hz), 7.32–7.40 (m, 3H), 7.56–7.63 (m,
H), 7.65–7.71 (m, 1H), 7.74 (dd, 1H, J ¼ 8.8, 2.5 Hz), 7.86 (d, 1H,
J ¼ 2.5 Hz), 10.65 (s, 1H) ppm
H NMR (400 MHz, DMSO-d ) d ¼ 4.41–4.45 (m, 2H), 5.98–6.06
6
(
2
(
m, 1H), 6.70–6.74 (m, 1H), 6.90 (d, 1H, J ¼ 11.2 Hz), 7.32–7.38 (m,
H), 7.80 (dd, 1H, J ¼ 8.8, 2.6 Hz), 7.87 (d, 1H, J ¼ 2.6 Hz), 7.95–7.97
m, 1H), 10.41 (s, 1H) ppm
1
3
13
1
.
C NMR (100 MHz, DMSO-d ) d ¼ 51.1, 116.2 (d, J ¼ 21.7 Hz),
C NMR (100 MHz, DMSO-d ) d ¼ 51.2, 112.3, 115.2, 120.9,
6
6
1
21.0, 121.4, 121.5, 122.8, 124.6 (d, J ¼ 5.5 Hz), 124.7 (d, 122.0, 122.2, 122.6, 128.3, 131.4, 137.3, 142.7, 146.0,
J ¼ 6.3 Hz), 128.5, 129.9 (d, J ¼ 2.6 Hz), 131.4, 132.8 (d, 147.2, 156.3 ppm
þ
J ¼ 8.5 Hz), 137.5, 142.8, 159.9 (d, J ¼ 249 Hz), 163.0 ppm
HRMS (ESI) [M þ H] : m/z calcd for (C H NO S) 306.0436
1
4
12
5
þ
2
.
HRMS (ESI) [M þ H] : m/z calcd for (C H FNO S) 334.0549 Found 306.0463.
16
13
4
Found 334.0554.
2.2. CA inhibitory assay
N-(2,2-Dioxido-3H-1,2-benzoxathiepin-7-yl)-2-(trifluoromethyl)ben-
zamide (15).
An applied photophysics stopped-flow instrument has been used
2
0
Compound 15 was prepared accord- for assaying the CA catalysed CO
ing to the general procedure from (at a concentration of 0.2 mM) was used as indicator, working at
amino derivative
(150 mg; the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5)
0.71 mmol), 2-(trifluoromethyl)ben- as buffer and 20 mM Na SO (for maintaining constant the ionic
zoyl chloride (115 mL; 0.78 mmol) strength), following the initial rates of the CA-catalysed CO hydra-
concentrations
2
hydration activity . Phenol red
H
N
7
CF3
O
2
4
SO2
O
2
and Et
Mp 192–193 C.
3
N (110 mL; 0.78 mmol) as white solid (236 mg; 87%). tion reaction for a period of 10 ꢂ 100 s. The CO
2
ꢀ
ranged from 1.7 to 17 mM for the determination of the kinetic
ꢂ1
IR (film, cm ) ꢀmax¼ 3195 (N–H), 1666 (C ¼ O), 1377 (S ¼ O), parameters and inhibition constants. For each inhibitor, at least six
316 (S ¼ O), 1164 (S ¼ O); traces of the initial 5 ꢂ 10% of the reaction have been used for
1

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
653
SO Cl
2
3
O N
O N
O N
2
2
2
O
(
i)
(ii)
OH
OH
O S
O O
1
2
4
(
iii) 5
H
N
R
H N
O N
2
2
(
v)
(iv)
O
SO2
SO2
SO2
O
O
O
8-17
7
6
R=
Me
;
;
Me
;
Br ;
N
N
8
(70%)
9 (72%)
10 (73%)
11 (59%)
I
Br
F
F3C
Ph
Cl
Cl
Ru
PCy3
;
;
;
;
;
1
2 (88%)
13 (82%)
14 (79%)
15 (87%)
5
.
S
O
1
6 (81%)
17 (81%)
ꢀ
ꢀ
Scheme 1. Reagents and conditions: (i) MePPh
3
Br, tBuOK, THF, RT, 18h, 65%; (ii) Net
3
, CH
2
Cl
2
, 0 C to RT, 4 h, 57%; (iii) 5, toluene, 70 C, 4 h, 96%; (iv) Fe, AcOH,
ꢀ
ꢀ
EtOH, H
2
O, 75 C, 1 h, 98%; (v) RCOCl, Net
3 2
, CH Cl
2
, 0 C to RT, 4 h.
determining the initial velocity. The uncatalysed rates were deter-
Table 1. Inhibition data of human CA isoforms CA I, II, IX and XII with 3H-1,2-
mined in the same manner and subtracted from the total benzoxathiepines 2,2-dioxide 8–17 using acetazolamide (AAZ) as a standard
drug.
observed rates. Stock solutions of inhibitor (0.1 mM) were pre-
H
pared in distilled ꢂ deionised water, and dilutions up to 0.01 nM
R
N
were done thereafter with the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 6 h at room temperature
prior to assay in order to allow for the formation of the E ꢂ I com-
plex. The inhibition constants were obtained by nonlinear least-
squares methods using PRISM 3 and the Cheng ꢂ Prusoff equa-
O
S
O
O
O
8
-17
21–23
a,b
tion, as reported earlier
, and represent the mean from at least
I
K (nM)
three different determinations. All CA isoforms were recombinant
21,24
Cmpd
R
hCA I
hCA II
hCA IX
hCA XII
ones obtained in-house as reported earlier
.
8
9
CH
C H
6 5
3
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
250
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
>100 mM
12
61.8
208.6
83.0
353.3
45.4
66.8
74.6
19.7
177.5
210.1
25
162.5
370.1
309.3
140.7
643.7
96.2
40.3
8.7
73.2
134.4
5.7
1
1
1
1
1
0
1
2
3
4
4-CH
4-Br-C
2-I-C
2-Br-C
2-F-C
2-CF
3 6 4
-C H
6
3
. Results and discussion
H
4
4
6
H
3.1. Chemistry
6
H
4
6
H
4
Starting from the benzaldehyde derivative 1, the synthesis of the
1
15
3
-C
6
H
4
key intermediate 7 was reported earlier by our groups . Briefly,
1
1
6
7
thien-2-yl
furan-2-yl
–
the synthesis of 7-amino-3H-1,2-benzoxathiepine 2,2-dioxide (7)
was started with a Wittig reaction in which 5-nitro-salicylic alde- AAZ
hyde 1 was converted to the corresponding mono-olefin 2 in 65%
a
Mean from three different assays, by a stopped flow technique (errors were in
yield (Scheme 1). Treatment of compound 2 with allyl sulphonyl the range of ±5–10% of the reported values).
b
Incubation time 6 h.
chloride (3) provided the bisolefin 4 in 65% yield. In the next
step, the olefin metathesis reaction with Ru-catalyst 5 was
employed, leading to the conversion of compound 4 to 7-nitro- the corresponding amine 7 in nearly quantitative yield (98%). The
3
H-1,2-benzoxathiepine 2,2-dioxide 6 in 96% yield. The nitro key intermediate 7 was subsequently reacted with a series of acyl
derivative 6 was thereafter reduced with iron in acidic medium to chlorides to afford the desired compounds 8–17 in good to

6
54
A. PUSTENKO ET AL.
excellent yields (see Experimental for details). The nature of moi- Basic Research [project grant 18–515-76001] under ERA.Net RUS
eties R was chosen in such a way to assure chemical diversity. plus joint programme grant RUS_ST2017-309.
Apart R ¼ Me in compound 8, the remaining derivatives 9–17
incorporated aromatic or heterocyclic moieties, such as phenyl, 2-
or 4-substituted phenyls, thienyl and furyl. We found out in previ-
1–3
ORCID
ous papers
that aryl or hetaryl moieties on the sulfocoumarin,
6
homosulfocoumarin or coumarin ring systems lead to com-
pounds with an effective inhibition profile against CA isoforms of
pharmacologic interest, such as the tumour-associated ones CA IX
and XII.
Claudiu T. Supuran
http://orcid.org/0000-0003-4262-0323
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4
. Conclusions
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Disclosure statement
The author(s) do not declare any conflict of interest.
2
010;20:7255–8; (b) Maresca A, Supuran CT. Coumarins
incorporating hydroxy- and chloro-moieties selectively
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