Synthesis and Biological Evaluation of 4-Sulfamoylphenyl/Sulfocoumarin Carboxamides as Selective Inhibitors of Carbonic Anhydrase Isoforms hCA II, IX, and XII

Article abstract of DOI:10.1002/cmdc.201800180

With the aim to develop potent and selective human carbonic anhydrase inhibitors (hCAIs), we synthesized 4-sulfamoylphenyl/sulfocoumarin benzamides (series 5 a–r and series 7 a–q) and evaluated their inhibition profiles against five isoforms of the zinc-containing human carbonic anhydrase (hCA, EC 4.2.1.1): cytosolic hCA I and II, and the transmembrane isozymes hCA IV, IX, and XII. Compounds 5 a–r were found to selectively inhibit hCA II in the nanomolar range, while being less effective against the other hCA isoforms. As noted from the literature, sulfocoumarin (1,2-benzoxathiine 2,2-dioxide) acts as a “prodrug” inhibitor and is hydrolyzed by the esterase activity of hCA to form 2-hydroxyphenylvinylsulfonic acid, which thereafter binds to the enzyme in a manner similar to that of coumarins and sulfoxocoumarins. All these sulfocoumarins (compounds 7 a–q) were found to be very weak or ineffective as inhibitors of the housekeeping off-target hCA isoforms I and II, and effectively inhibited the transmembrane tumor-associated isoforms IX and XII in the high nanomolar to micromolar ranges. Further structural modifications of these molecules could be useful for the development of effective hCA inhibitors used for the treatment of glaucoma, epilepsy, and cancer.

Full text of DOI:10.1002/cmdc.201800180

Accepted Article
Title: Synthesis and Biological Evaluation of 4-sulfamoylphenyl/
sulfocoumarin carboxamides as selective inhibitors of carbonic
anhydrase isoforms hCA II, IX and XII
Authors: Srinivas Angapelly, Andrea Angeli, Arbaj Jabbar Khan,
Posa Venkata SriRamya, Claudiu Trandafir Supuran, and
Mohammed Arifuddin
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201800180
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800180
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Synthesis and Biological Evaluation of 4-
sulfamoylphenyl/sulfocoumarin carboxamides as selective
inhibitors of carbonic anhydrase isoforms hCA II, IX and XII
Srinivas Angapelly,[a] Andrea Angeli,[b] Arbaj Jabbar Khan,[a] P. V. Sri Ramya,[a] Claudiu T. Supuran,*[b]
Mohammed Arifuddin,*[a]
[a]
Srinivas Angapelly,[a] Arbaj Jabbar Khan,[a] Posa Venkata Sri Ramya,[a] and Dr. Mohammed Arifuddin*[a]
Department of Medicinal Chemistry
National Institute of Pharmaceutical Education & Research (NIPER)
Hyderabad-500037, India
E-mail: arif@niperhyd.ac.in
[b]
Andrea Angeli,[b] and Prof. Claudiu T. Supuran*[b]
NEUROFARBA Dept., Sezione di Scienze Farmaceutiche
Università degli Studi di Firenze
Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
E-mail: claudiu.supuran@unifi.it
Abstract: With the aim to develop potent and selective human
carbonic anhydrase inhibitors (hCAIs), we synthesized 4-
sulfamoylphenyl/sulfocoumarin benzamides (5a–r and 7a–q) and
evaluated for their inhibition profiles against five isoforms of zinc
enzyme carbonic anhydrase (CA, EC 4.2.1.1), the cytosolic CA I and
II as well as transmembrane isozymes CA IV, IX and XII.
Compounds 5a–r selectively inhibited the isoform hCA II in
nanomolar range, being less effective against the other isoforms. As
noticeable from the literature, sulfocoumarin (1,2-benzoxathiine 2,2-
dioxide) act as “prodrug” inhibitor and get hydrolysed by the esterase
CA activity to form 2-hydroxyphenyl-vinylsulfonic acid, which
thereafter bind to the enzyme in a similar fashion like coumarins and
sulfoxocoumarins. All these sulfocoumarins (7a–q) were very weak
or ineffective as inhibitors of the housekeeping, off-target isoforms
process in all life forms, since many biochemical transformations
are tightly regulated by pH.[4]
In humans, 15 distinctive CA isozymes, all belonging to the α-
class have been identified to date, and displaying differences in
molecular features, oligomeric arrangement, tissue-specific
expression, kinetic properties, physiological/pathological roles,
and susceptibility to inhibitors.[5a] Based on their subcellular
localization, these isoforms can be divided as follows: cytosolic
(CA I, CA II, CA III, CA VII, CA XIII), mitochondrial matrix
isoforms (CA VA, CA VB), transmembrane isoforms (CA IV, CA
IX, CA XII, CA XIV) and a secreted isozyme (CA VI). Several
studies demonstrated vital roles of CAs in some of physiological
pathways such as intracellular and extracellular pH homeostasis,
modulation of neuronal transmission, cell proliferation and
differentiation and several biochemical pathways where either
CO2 or bicarbonate is required. Immense experimental evidence
also suggested that the dysregulation (or aberrant expression) of
these enzymes in one or more tissues leads to various
pathological conditions and thus are interesting therapeutic
targets for the treatment of glaucoma, obesity, epilepsy and
cancer.[5b-c-8]
CA
I and II, and effectively inhibited transmembrane, tumor
associated isoforms hCA IX and XII in high nanomolar to micromolar
range. Further structural modifications of these molecules could be
useful for the development of effective CA inhibitors that will be used
for the treatment of glaucoma, epilepsy and cancer.
Carbonic anhydrases (CAs, EC 4.2.1.1) are superfamily of zinc
dependent metalloenzymes, present in prokaryotes and
eukaryotes, and catalyses the reversible hydration of carbon
dioxide (a central element for life on this planet) to afford
CA II is one of the most active and highly abundant cytosolic
isoform, expressed virtually in every human tissue and organ.
Indeed, dysregulation of this isoform in one or more tissues has
been an established drug target for a multitude of diseases,
such as glaucoma, edema, epilepsy, some forms of cancer, in
which CA II was observed to be over-expressed alone or
together with the other isoforms such as CA IX and XII, as in
case of acute mountain sickness (AMS) and, apparently,
atherosclerosis and osteoporosis.[9-11] Moreover, CA II is a target
for imaging in diverse pathological conditions, in organs where
the enzyme is present, such as the brain and cerebrospinal fluid,
or the gastrointestinal tract, etc.[12]
monohydrogen carbonate and protons by using
a metal
hydroxide nucleophilic mechanism.[1] However, the hydration of
CO2 is particularly slow at pH values of 7.5 or lower, which is
usually the physiologic pH value in many tissues and organisms,
and on the other hand, very effective at higher pH values, being
instantaneous at pH values over 12.[2] So far, seven genetically
distinct families of CAs were described in detail, i.e., the human
α-, the β-, γ-, δ-, ζ-, η- and the recently reported θ-CAs.[3] These
CA families differ in their preference for the metal ion being
present within the active site cavity for performing the catalysis
as well as in the three-dimensional fold of the protein backbone,
constituting a paradigmatic example of convergent Darwinian
evolution at the molecular level. These enzymes not only
converts the high amounts of CO2 formed in the metabolic
processes, transforming it into water soluble ions, but also
manage the possible acid–base equilibria connected to this
reaction. In fact, the regulation of pH is a unique physiological
In the recent years, among the different mammalian isoforms,
the hCA VII, IX, and XIV became popular druggable targets for
inhibitor development. hCA VII is one of the least explored
cytosolic CA isozyme; it presents a limited distribution, being
mainly expressed in some parts of the brain (hippocampus,
cortex, and thalamus regions) where it is considered involved in
1
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triggering neuronal excitation and seizures. In recent times, the
role of hCA VII in neuropathic pain control was projected. This
could represent an intriguing pharmacological mechanism for
the design of new pain killers useful for biomedical
applications.[13] In the last decade, CA IX has garnered more
interest because of its restricted expression in normal tissues,
whereas it is over expressed in many solid tumors, its apparent
role in pH control, cell proliferation and migration, cell adhesion,
and tumorigenesis.[14-16] The expression of hCA IX is strongly
induced by hypoxia via the hypoxia inducible factor-1 (HIF-1), a
transcription factor. Therefore, targeting this specific isoform
could be beneficial for the design of new therapeutic and
diagnostic tools in cancer treatment. Indeed, several monoclonal
antibodies (for example, Girentuximab, and 177Lu-cG250)
targeting CA IX are in phase III clinical trial for the treatment of
solid tumors (or for their imaging), while some small molecule
inhibitors [SLC-0111(V) and DTP-348 (VI)] are in various stages
of clinical development.[17-18a-b] Moreover, recently reported
sulfocoumarin derivatives (for example, VII) selectively target
tumor associated isoforms hCA IX and XII.[18c] hCA XIV is a
transmembrane isoform with the active site aligned
extracellularly; it is strongly expressed in neurons and axons in
the human brain and murine, where it seems to play a vital role
in modulating excitatory synaptic transmission.[19-20] Some of the
drugs/drug candidates with potent CA inhibitory activity are
depicted in Figure 1. Nevertheless, the CA diffuse localization in
many tissues and organs restricts potential clinical applications.
So the development of CA inhibitors (CAIs) possessing high
potency and selectivity against some specific isoforms
represents an attractive strategy to obtain valuable
pharmacological tools, and thus avoiding side effects and
improving therapeutic safety.
Figure 1: Some of the drugs/drug candidates with potent CA inhibitory activity.
Figure 2: CA inhibition mechanisms: A. Zinc-binders; B. Compounds which anchor to the metal ion coordinated water/hydroxide ion; C. Occlusion of the active
site entrance. D. Out of the active site binding.
Till date so many novel chemotypes have been investigated as
potent CA inhibitors, which exhibit their CA inhibitory activity via
diverse inhibition mechanisms (Figure 2). Among them,
sulfonamides and their bioisosters, and recently discovered
boronic acids, hydroxamates and dithiocarbamates directly bind
to the Zn2+ metal ion of the active site (Fig. 2A). Indeed,
“sulfocoumarins” (bioisosters of coumarins) are recently reported
as potent and isoform-selective “prodrug” CAIs.[18c,26]
Sulfocoumarins (1) initially undergoes hydrolysis by the CA-
mediated esterase activity to form (z)-vinyl sulfonic acid (2z),
which spontaneously generates a stable (E)-vinyl sulfonic acid
species upon geometrical isomerisation (Figure 3A) and thus,
the sulfocoumarin derivatives have been considered as de facto
CA inhibitors. Kinetic as well as X-ray crystallographic
experiments on CA II adducts revealed that the sulfonic acid
moiety did not make any interactions with the amino acid
residues within the active site except anchoring to the zinc-
coordinated water/hydroxide ion (Figure 3B).[5a,21] But the −OH
2
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10.1002/cmdc.201800180
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COMMUNICATION
group ortho to the ethylene sulfonate moiety has only
participated in hydrogen bonding with the hydroxyl of Thr200 (of
3.3 A°) and through a bridging water molecule, with the carbonyl
oxygen of Pro201 (of 2.7 A°). This type of phenomenon has
been never observed in case of widely investigated sulfonamide
inhibitors. In continuation of our earlier works,[22-23] herein we
focused on the synthesis of 4-sulfamoylphenyl/sulfocoumarin
carboxamides to evaluate their CA inhibitory activity against
various CA isozymes.
As outlined in Scheme 1, the synthesis of compounds 5a–r and
7a–r starts from para-substituted benzoic acids (1a–e), which
were converted into 3-(chlorosulfonyl)benzoic acids (2a–e) by
the reaction with chlorosulfonic acid at 110 °C (70-85% yield).
The sulfonyl chlorides (2a–e) were then reacted with
a
secondary amine to afford the respective 3-sulfamoyl benzoic
acids (3a–e). Next, these compounds underwent acid-amine
coupling reaction with sulfanilamide (4) and 6-amino
sulfocoumarin (6)[24] in the presence of EDCI and HOBT in DMF
to furnish the desired 4-sulfamoylphenyl/sulfocoumarin
carboxamide derivatives (5a–r and 7a–q) respectively, in good
yields (50-70%). Moreover, all the synthesized analogs were
well characterized by various spectroscopic methods i.e., NMR
(1H and 13C) and HRMS.
Figure 3: CA inhibition mechanism of sulfocoumarin. (A) The sulfocoumarin 1
undergoes an enzyme-mediated hydrolysis with the formation of E-2-
hydroxyphenyl-ω-ethenylsulfonic acid.[26c] (B) Binding of sulfocoumarin 3 within
the active site of the CA II/IX mimic, determined by X-ray crystallography (PDB
ID: 4BCW).[26c]
Scheme 1: Synthesis of 4-sulfamoylphenyl/sulfocoumarin carboxamide derivatives. Reagents and conditions: a) HSO3Cl, 110 °C, 5-8 h, 70-85%; b) 2° Amine (R1),
DCM, rt, 8-14 h, 80-90%; c) EDCI, HOBT, R2NH2 (4 and 6), rt, 16-24 h, 50-70%.
Table 1: List of the synthesized compounds (5a-r and 7a-q)
Compound
R
R1
Compound
R
R1
5a
5b
5c
5d
5e
5f
H
H
Morpholine
Cis-2,6-dimethylmorpholine
Piperidine
7a
7b
7c
7d
7e
7f
H
H
Morpholine
Cis-2,6-dimethylmorpholine
Pyrrolidine
H
H
H
Pyrrolidine
OCH3
OCH3
OCH3
OCH3
Cl
Morpholine
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
Morpholine
Cis-2,6-dimethylmorpholine
Pyrrolidine
Cis-2,6-dimethylmorpholine
Piperidine
5g
5h
5i
7g
7h
7i
Thiomorpholine
Pyrrolidine
Morpholine
Thiomorpholine
Morpholine
Cl
Cis-2,6-dimethylmorpholine
Piperidine
5j
7j
Cl
3
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5k
5l
Cl
Cl
Cis-2,6-dimethylmorpholine
Pyrrolidine
7k
7l
F
F
Morpholine
Cis-2,6-dimethylmorpholine
Piperidine
5m
5n
5o
5p
5q
5r
F
Morpholine
7m
7n
7o
7p
7q
F
F
Cis-2,6-dimethylmorpholine
Piperidine
F
Pyrrolidine
F
CH3
CH3
CH3
Cis-2,6-dimethylmorpholine
Piperidine
CH3
CH3
CH3
Cis-2,6-dimethylmorpholine
Piperidine
Pyrrolidine
Pyrrolidine
7d
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
250
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
12.1
370.3
3360.0
3482.0
3250.0
3186.0
3221.6
1646.0
99.4
2326.9
2708.0
4913.0
4380.3
5901.0
6038.8
6696.5
7461.5
5772.7
36.9
>27
>2.9
>2.8
>3.0
>3.1
>3.1
>6.0
>100
>3.4
>85.1
>83.5
>94.6
>44.4
>26.1
9.6
>4.2
>3.6
>2.0
>2.2
>1.7
>1.6
>1.5
>1.3
>1.7
>271
>36.5
>326
>3.3
>23.4
43.8
>27
>2.9
>2.8
>3.0
>3.1
>3.1
>6.0
>100
>3.4
>85.1
>83.5
>94.6
>44.4
>26.1
0.4
>4.2
>3.6
>2.0
>2.2
>1.7
>1.6
>1.5
>1.3
>1.7
>271
>36.5
>326
>3.3
>23.4
2.1
The newly synthesized compounds (5a–r and 7a–q) were tested
for their ability to inhibit a panel of representative isoforms of
human CAs (I, II, IV, IX, and XII, Table 2 and 3). Catalytic
activities were measured by a stopped-flow assay,[25a] and
acetazolamide was used as a standard.
7e
7f
7g
7h
7i
Table 2: Inhibitory potency data against isoforms hCA I, II, IV and IX along
with selectivity ratio for inhibition of isoform hCA II over hCA I, IV and IX for
compounds 5a-r
7j
7k
7l
KI (nM)a
hCA II
17.1
37.5
8.6
Selectivity ratiob
IV/II
2896.8
117.5
119.7
105.7
224.9
383.0
25.8
Compd
5a
hCA I
195.9
646.4
88.0
hCA IV
6453.2
5006.2
4890.6
5051.0
5253.8
4198.3
768.4
hCA IX
1957.5
1690.0
431.5
I/II
IX/II
114.4
45.06
568.6
652.5
373.0
564.0
68.1
46.8
303.1
619.1
74.4
183.4
483.1
56.9
30.36
0.7
7m
7n
7o
7p
7q
AAZ
11.4
17.2
10.2
61.0
9.9
377.3
133.4
568.6
953.0
632.9
666.3
15.5
273.4
30.6
5b
5c
2960.1
426.2
5.7
5d
5e
323.5
82.7
5.3
3458.5
3096.0
3553.5
3361.0
4314.0
4092.5
4024.5
3561.0
4365.0
3817.0
296.0
8.3
5f
280.2
820.9
943.6
421.4
87.2
6.3
44.4
16.7
10.2
31.2
13.4
11..1
27.1
85.8
8.7
a Mean from 3 different assays, by a stopped flow technique (errors were in the
range of  5-10 % of the reported values). b Selectivity as determined by the
ratio of Kis for hCA isozyme relative to hCA IX and hCA XII.
5g
5h
5i
49.3
92.1
13.5
6.5
3891.6
3327.1
539.9
42.2
The following structure activity relationship (SAR) was observed
by investigating the inhibition data of Table 2 and 3.
246.4
83.0
5j
5k
529.8
645.8
677.5
45.5
47.8
23.8
7.9
470.0
9.8
i) Most of the synthesized compounds of sulfonamide class
(5a–r) did not exhibit potent inhibitory activity for isoform
hCA I, which is a slow cytosolic and off-target isozyme for
antiepileptics.[25b] Among all these derivatives, 3-(piperidin-1-
ylsulfonyl) (5c), 4-methoxy-3-morpholinosulfonyl (5e), 4-
5l
538.7
22.6
5m
5n
5o
5p
5q
5r
840.2
106.3
1552.3
1389.8
20.14
15.9
5.2
8072.3
8756.0
8344.6
6651.7
5788.3
74
434.2
5379.5
5997.1
169.7
250
6.3
191.3
68.9
12.9
14.3
3.5
chloro-3-(morpholinosulfonyl)
(5j),
4-fluoro-3-(cis-2,6-
dimethylmorpholinosulfonyl) (5n) derivatives displayed
medium inhibitory potency (KI 45.5-88.0 nM) against isoform
hCA I, whereas the compounds of sulfocoumarin class (7a–
q) were very weak or ineffective inhibitors of this isoform
(Table 3).
414.3
417.8
48.5
12.1
294.9
260.2
0.6
282.5
119.3
6.1
5.8
AAZ
25.8
20.6
2.1
a Mean from 3 different assays, by a stopped flow technique (errors were in the
range of  5-10 % of the reported values). b Selectivity as determined by the
ratio of Kis for hCA isozyme relative to hCA I, IV and hCA IX.
ii) The cytosolic and physiologically dominant isoform hCA II,
was strongly inhibited by the majority of the sulfonamides
(5a–r) investigated here, with KI values spanning between
5.1 and 417.8 nM. Compounds 5c–f, 5j, 5m–o inhibited hCA
II more effectively, being superior to AAZ (KI values ranging
between 5.1 and 8.6 nM versus 12.1 nM for AAZ).
Analogues 5a–b, 5g, 5i, 5k–l, and 5r displayed slightly
weaker activity (KI values of 13.5−48.5 nM) in comparison to
the standard drug, whereas the rest of the molecules 5h,
5p–q showed high nanomolar inhibitory activity (KI values of
92.1-417.8 nM). SAR studies revealed that the methyl
substitution on the phenyl ring lessen the potency of
Table 3: Inhibitory potency data against isoforms hCA I, II, IX and XII along
with selectivity ratio for inhibition of isoforms hCA IX and XII over hCA I and II
for compounds 7a-q
KI (nM)a
hCA II
Selectivity ratiob
Compd
7a
hCA I
>10000
>10000
>10000
hCA IX
292.4
hCA XII
258.8
I/IX
I/XII
II/IX
>34.1
>7.5
II/XII
>38.6
>37.1
>17.8
>10000
>10000
>10000
>34.1
>7.5
>38.6
>37.1
>17.8
7b
1331.6
309.1
269.2
7c
561.5
>32.3
>32.3
4
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inhibition, for example analogues 5b, 5c and 5d are 11, 48
IX and XII: these derivatives bind at the entrance of the
active site cavity which is the most variable region between
the many CA isoforms known to date.
and
9 times more potent respectively, than methyl
substituted compounds i.e., 5p, 5q and 5r. Among the 3-
(piperidin-1-ylsulfonyl) derivatives, compound 5o carrying
electron withdrawing group (fluoro) at C-4 position showed
better inhibitory activity than the –OCH3 (5g) and methyl (5q)
substituted analogues. Compounds belonging to the class of
sulfocoumarins (7a–q) were not active against this isoform
(Table 3).
In conclusion, two series of 4-sulfamoylphenyl/sulfocoumarin
carboxamide derivatives (5a‒r, 7a‒q) have been synthesized
successfully and evaluated in-vitro for their CA inhibitory profile
against cytosolic isoforms (hCA I and II) and membrane-bound
isoforms (hCA IV, IX and XII). From the in-vitro assay results it
was observed that compounds 5a‒r displayed moderate
inhibitory activity against hCA I (KI 45.5˗5997.1 nM) and more
selectivity towards hCA II (KI 5.2˗417.8 nM), and compounds
7a‒q are very weak or ineffective against isoforms hCA I and II.
Moreover, sulfonamide class of compounds (5a‒r) exhibited
iii) hCA IV, which is a membrane-bound isozyme majorly
expressed in the eye, being involved in glaucoma and
retinitis pigmentosa among other CA-related pathologies.
Most of the investigated compounds (5a–r) exhibited
inhibitory activity in micro-molar range (KI 3327.1˗8756.0 nM), moderate CA inhibition against isoforms hCA IV and IX (KI
whereas, compounds 5g, 5j–m only showed nanomolar
inhibition (KI 470.0˗840.2 nM) against this isoform. From the
activity results (Table 2), it was clearly demonstrated that the
compounds bearing chloro group (5k–n) showed better CA
inhibitory activity in comparison to the other substituted
analogues.
191.3˗8756.0 nM) and sulfocoumarins (7a‒q) selectively
inhibited membrane-bound, tumor associated isoforms hCA IX
and XII. Among all the investigated compounds here, analogue
5j carrying 4-chloro-3-(morpholinosulfonyl) showed better
inhibitory profile against isoforms hCA II and IV, being involved
in ocular disorders and 7m and 7o displayed potent inhibitory
activity against both the isoforms CA IX and XII (KI = 30.6-117.5
nM). Overall, 4-sulfamoylphenyl/sulfocoumarin carboxamides
analogues described in the present work indicated exciting
possibilities of developing potent hCA inhibitors that target
ocular disorders and cancer via their structural modifications.
iv) The transmembrane and tumor associated isoform hCA IX,
was inhibited by both sulfonamide (5a–r) and sulfocoumarin
(7a–q) analogues, in medium nanomolar to micromolar
range, with KI values of 99.4 nM−4092.5 nM. Among all the
sulfonamides, compound 5o possessing 4-fluoro-3-
(piperidin-1-ylsulfonyl) moiety was the best inhibitor against
this isoform with KI value 191.0 nM. In case of C-4 fluoro
substituted compounds (5m, 5n and 5o), when cis-2,6-
dimethylmorpholine (5n) and piperidine (5c) was replaced by
the morpholine ring (5m) the activity decreased to 3817.0
nM. Moreover, when −OCH3 was replaced with methyl group,
the activity was increased significantly from micromolar to
nanomolar range. In case of all sulfocoumarins, analogue 7k
showed prominent hCA IX inhibitory activity with KI value of
99.4 nM. Compounds with fluoro and methyl substitution
showed nanomolar activity (KI = 99.4−383.0 nM) except for
Experimental
All solvents were purified and dried using standard methods prior to use.
Commercially available reagents were used without further purification.
All reactions involving air- or moisture-sensitive compounds were
performed under a nitrogen atmosphere using dried glassware and
syringe techniques to transfer solutions. Analytical thin-layer
chromatography (TLC) was carried out on Merck silica gel 60 F-254
aluminium plates. Melting points were determined on Stuart digital
melting-point apparatus/SMP 30 in open capillary tubes and are
uncorrected. Nuclear magnetic resonance (1H-NMR, 13C-NMR) spectra
were recorded using an Avance Brucker 500 MHz, 125 MHz
spectrometer in DMSO-d6. Chemical shifts are reported in parts per
million (ppm) with TMS as an internal reference, and the coupling
constants (J) are expressed in hertz (Hz). Splitting patterns are denoted
as follows: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of
doublets. HRMS were determined with Agilent QTOF mass spectrometer
6540 series instrument and were performed in the ESI techniques at 70
eV.
compound 7l (KI
=
2896.8 nM) where cis-2,6-
dimethylmorpholine ring could be the probable reason for the
drop in the inhibition potency. The rest of the molecules such
as 7b, 7e‒j did not inhibit this isoform effectively.
v) The other transmembrane, tumor-associated human isoform,
hCA XII, was also inhibited selectively by all the tested
sulfocoumarin carboxamides in nanomolar-micromolar range.
Sulfocoumarins bearing 4-fluoro-3-(piperidin-1-ylsulfonyl)
(7m), 4-methyl-3-(cis-2,6-dimethylmorpholinosulfonyl) (7o)
have more affinity for this isoform with KI values of 30.6 nM
and 36.9 nM and compounds 7a‒c and 7q exhibited high
nanomolar hCA inhibitory activity, whereas the remaining
molecules inhibited this isoform in the micromolar range (KI
of 2326.9-7461.5 nM).
Synthesis of 3-(chlorosulfonyl)benzoic acid derivatives (2a-
e).
Benzoic acid (1a-e) (5g, 40.9 mmol) was added in portion wise
to chlorosulfonic acid (13.6 mL, 204.5 mmol) under stirring at 0
°C and then stirred at 110 °C for 5-8 h. The reaction was
monitored by TLC; after completion of the reaction, the reaction
mixture was cooled to RT and was then poured into vigorously
stirred crushed-ice (200 g) and the obtained solid was filtered,
washed with water (50 mL) and dried under vacuum to obtain
desired intermediates (2a-e), as white solid in 70-85% yields,
which were carried forward without further purification.
vi) From the above results, it has been demonstrated that the
sulfocoumarin derivatives are mechanism based CA
inhibitors (Figure 2B, 2C and 3), a phenomenon never
observed for the main class of CA inhibitors such as
sulfonamides, and their isosteres (sulfamates, sulfamides,
etc.).[18c,26] This could be the probable reason for the
selectivity profile of compounds 7a‒q against hCA isoforms
Synthesis of 3-sulfamoylbenzoic acids (3a-e):
5
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10.1002/cmdc.201800180
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COMMUNICATION
325‒332; (c) C. T. Supuran, J. Enzyme. Inhib. Med. Chem. 2012, 27,
759‒772.
A solution of substituted 3-(chlorosulfonyl) acid (2a-e) (0.5 g,
2.28 mmol) in dichloromethane (10 mL) was treated with
secondary amine (9.13 mmol,). After 8-14 h, the reaction was
quenched with 10% HCI and extracted with EtOAc. The organics
were washed with brine solution and then dried over Na2SO4.
Organic layer were then removed under reduced pressure to
obtain 3a-e in yields 80-90%.
2. (a) C. T. Supuran, Biochem. J. 2016, 473, 2023‒2032; (b) C. T. Supuran,
J. Enzyme Inhib. Med. Chem. 2013, 28, 229‒230.
3. (a) G. De Simone, C. T. Supuran, J. Inorg. Biochem. 2012, 111, 117‒129;
(b) S. Kikutani, K. Nakajima, C. Nagasato, Y. Tsuji, A. Miyatake, Y.
Matsuda, Proc. Natl Acad. Sci. U.S.A. 2016, 113, 9828‒9833.
4. (a) S. Del Prete, D. Vullo, G. M. Fisher, K. T. Andrews, S. A. Poulsen, C.
Capasso, C. T. Supuran, Bioorg. Med. Chem. Lett. 2014, 24, 4389‒4396.
5. (a) V. Alterio, A. Di Fiore, K. D’Ambrosio, C. T. Supuran, G. De
Simone, Chem. Rev. 2012, 112, 4421‒4468; (b) A. Scozzafava, L.
Menabuoni, F. Mincione, C. T. Supuran, J. Med. Chem. 2002, 45,
1466‒1476; (c) F. Carta, C. T. Supuran, A. Scozzafava, Expert Opin.
Ther. Pat. 2012, 22, 79‒88.
Synthesis
of
4-sulfamoylphenyl/sulfocoumarin
carboxamides (5a-r and 7a-q):
To a stirred solution of substituted 3-sulfamoylbenzoic acids 3a-e
(0.5 mmol) in dry DMF (5 mL), EDCI (0.55 mmol), HOBt (0.55
mmol), were added and allowed to stir for 30 min at room
temperature. Later on, 4-sulfanilamide/sulfocoumarin (0.55
mmol) was added to the above reaction mixture, and was stirred
at rt until starting material was consumed (TLC monitoring).
Then the reaction mixture was quenched with ice; the
precipitated products were filtered, and finally washed with cold
H2O. The resulting solids were recrystallized from aqueous
ethanol.
6. (a) E. Masini, F. Carta, A. Scozzafava, C. T. Supuran, Expert Opin. Ther.
Pat. 2013, 23, 705‒716; (b) F. Carta, C. T. Supuran, Expert Opin. Ther.
Pat. 2013, 23, 681‒691; (c) C. Capasso, C. T. Supuran, Expert Opin.
Ther. Pat. 2013, 23, 693-704.
7. (a) A. Scozzafava, C. T. Supuran, F. Carta, Expert Opin. Ther. Pat. 2013,
23, 725‒735; (b) A. Di Fiore, G. De Simone, V. Alterio, V. Riccio, J. Y.
Winum, F. Carta, C. T. Supuran, Org. Biomol. Chem. 2016, 14,
4853‒4858.
8. D. Neri, C. T. Supuran, Nat. Rev. Drug Discov. 2011, 10, 767‒777.
9. (a) F. Mincione, A. Scozzafava, C. T. Supuran, Curr. Pharm. Des. 2008,
14, 649‒654; (b) C. T. Supuran, Curr. Pharm. Des. 2008, 14, 641‒648.
10. (a) N. Hen, M. Bialer, B. Yagen, A. Maresca, M. Aggarwal, A. H. Robbins,
R. McKenna, A. Scozzafava, C. T. Supuran, J. Med. Chem. 2011, 54,
3977‒398; (b) G. De Simone, A. Scozzafava, C. T. Supuran, Chem. Biol.
Drug Des. 2009, 74, 317‒321; (c) B. Basnyat, J. H. Gertsch, E. W.
Johnson, F. Castro-Marin, Y. Inoue, C. Yeh, High Alt. Med. Biol. 2003, 4,
45‒52.
Carbonic anhydrase inhibition assay
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic/inhibition of
various CA isozymes.[25a] 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
Na2SO4 or NaClO4 (for maintaining constant the ionic
11. (a) K. Yoshiura, T. Nakaoka, T. Nishishita, K. Sato, A. Yamamoto, S.
Shimada, Clin. Cancer Res. 2005, 11, 8201-8207; (b) J. Haapasalo, K.
Nordfors, S. Jarvela, H. Bragge, I. Rantala, A. K. Parkkila, Neuro Oncol.
2007, 9, 308-313.
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 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 preincubated together for 10 min at
room temperature 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
earlier,[27] and represent the mean from at least three different
determinations. All CA isozymes used here were recombinant
proteins obtained as reported earlier by our group.[28-29]
12. (a) J. A. Detre, O. B. Samuels, D. C. Alsop, J. B. Gonzalez-At, S. E.
Kasner, E. C. Raps, J. Magn. Reson. Imaging, 1999, 10, 870-875; (b) R.
Takeuchi, H. Matsuda, Y. Yonekura, H. Sakahara, J. Konishi, J. Cereb.
Blood. Flow. Metab. 1997, 17, 1020-1032; (c) J. Pan, J. Lau, F. Mesak, N.
Hundal, M. Pourghiasian, Z. Liu, F. Bénard, S. Dedhar, C. T. Supuran, K.
S. Lin, J. Enzyme Inhib. Med. Chem. 2014, 29, 249-255.
13. (a) E. Ruusuvuori, H. Li, K. Huttu, J. M. Palva, S. Smirnov, C. Rivera, K.
Kaila, J. Voipio, J. Neurosci. 2004, 24, 2699‒2707; (b) M. Asiedu, M. H.
Ossipov, K. Kaila, T. J. Price, Pain 2010, 148, 302‒308.
14. (a) R. Del Giudice, D. M. Monti, E. Truppo, A. Arciello, C. T. Supuran, G.
De Simone, S. M. Monti, Biol. Chem. 2013, 394, 1343‒1348; (b) S.
Parkkila, H. Rajaniemi, A. K. Parkkila, J. Kivelä, A. Waheed, S.
Pastoreková, J. Pastorek, W. S. Sly, Proc. Natl Acad. Sci. U.S.A. 2000,
97, 2220‒2224.
15. (a) N. Robertson, C. Potter, A. L. Harris, Cancer Res. 2004, 64,
6160‒6165; (b) Y. Lou, P. C. McDonald, A. Oloumi, S. Chia, C. Ostlund,
A. Ahmadi, A. Kyle, U. auf dem Keller, S. Leung, D. Huntsman, B. Clarke,
B. W. Sutherland, D. Waterhouse, M. Bally, C. Roskelley, C. M.
Overall, A. Minchinton, F. Pacchiano, F. Carta, A. Scozzafava, N.
Touisni, J.-Y. Winum, C. T. Supuran, S. Dedhar, Cancer Res. 2011, 71,
3364‒3376.
Acknowledgement
16. (a) E. Švastová, A. Hulıková, M. Rafajová, M. Zat'ovičová, A.
́
SA and PVSR are thankful to Department of Pharmaceuticals,
Ministry of Chemicals and Fertilizers, Govt. of India, New Delhi
for the award of NIPER fellowship.
Gibadulinová, A. Casini, A. Cecchi, A. Scozzafava, C. T. Supuran, J.
Pastorek, S. Pastoreková, FEBS Lett. 2004, 577, 439‒445; (b) Y. Li, C.
Tu, H. Wang, D. N. Silverman, S. C. Frost, J. Biol. Chem. 2011, 286,
15789‒15796.
Keywords: 4-Sulfamoylphenyl/sulfocoumarin carboxamides, CA
inhibition, de facto inhibitors, glaucoma, cancer
17. (a) A. B. Stillebroer, O. C. Boerman, I. M. Desar, M. J. Boers-Sonderen, C.
M. van Herpen, J. F. Langenhuijsen, P. M. Smith-Jones, E. Oosterwijk, W.
J. Oyen, P. F. Mulders, Eur. Urol. 2013, 64, 478-485; (b) I. Bleumer, A.
Knuth, E. Oosterwijk, R. Hofmann, Z. Varga, C. B. H. W. Lamers, W. Kruit,
S. Melchior, C. Mala, S. Ullrich, P. De Mulder, Br. J. Cancer. 2004, 90,
985-990.
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COMMUNICATION
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7
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Entry for the Table of Contents
Novel series of 4-sulfamoylphenyl/sulfocoumarin carboxamides have been synthesized, and investigated as inhibitors of the
human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I, II, IV, IX and XII. Compounds 5a-r showed selectivity against
isoform hCA II, whereas, analogs 7a-q targeted tumor associated isozymes i.e., hCA IX and XII.
8
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