Benzamide-4-sulfonamides are effective human carbonic anhydrase I, II, VII, and IX inhibitors

Article abstract of DOI:10.3390/metabo8020037

A series of benzamides incorporating 4-sulfamoyl moieties were obtained by reacting 4-sulfamoyl benzoic acid with primary and secondary amines and amino acids. These sulfonamides were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA,

Full text of DOI:10.3390/metabo8020037

H
OH
metabolites
OH
Article
Benzamide-4-Sulfonamides Are Effective Human
Carbonic Anhydrase I, II, VII, and IX Inhibitors
Morteza Abdoli 1,2, Murat Bozdag 1, ID , Andrea Angeli 1 and Claudiu T. Supuran 1,
* *
ID
1
Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di
Firenze, Via U. Schiff 6, Sesto Fiorentino, 50019 Florence, Italy; mortezaabdoli1987@gmail.com (M.A.);
andrea.angeli@unifi.it (A.A.)
Department of Chemistry, Faculty of Science, Lorestan University, Khorramabad 6813833946, Iran
Correspondences: bozdag.murat@unifi.it (M.B.); claudiu.supuran@unifi.it (C.T.S.);
Tel.: +39-055-457-3666 (M.B.); +39-055-457-3729 (C.T.S.)
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 May 2018; Accepted: 30 May 2018; Published: 1 June 2018
Abstract: A series of benzamides incorporating 4-sulfamoyl moieties were obtained by reacting
4-sulfamoyl benzoic acid with primary and secondary amines and amino acids. These sulfonamides
were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The human
(h) isoforms hCA II, VII, and IX were inhibited in the low nanomolar or subnanomolar ranges, whereas
hCA I was slightly less sensitive to inhibition (KIs of 5.3–334 nM). The β- and γ-class CAs from
pathogenic bacteria and fungi, such as Vibrio cholerae and Malassezia globosa, were inhibited in the
micromolar range by the sulfonamides reported in the paper. The benzamide-4-sulfonamides are a
promising class of highly effective CA inhibitors.
Keywords: carbonic anhydrase; human isoform; sulfonamide; benzamide; pathogens
1. Introduction
Benzamides incorporating 3- or 4-sulfamoyl moieties, such as derivatives
investigated [ ] as inhibitors of the zinc metallo-enzyme carbonic anhydrase (CA, EC 4.2.1.1) [
in this study, in the search of agents with intraocular pressure lowering effects [ ]. The incorporation
of a wide range of amino acid (AA) or dipeptide AA moieties in molecules and led to enhanced
A
and
B
(Figure 1) were
1
,2
3–12
]
1,2
A
B
water solubility for topical administration within the eye. These compounds showed remarkable
in vitro inhibitory effects, assayed by an esterase method with 4-nitrophenyl acetate as substrate,
against isoforms hCA II and IV, involved in aqueous humor production within the eye [1–12].
SO2NH2
SO2NH2
Cl
H
N
AA
O
O
N
AA
H
(A)
(B)
Figure 1.
(A,B) Sulfonamides incorporating benzamide moieties, amino acid (AA) and dipeptide AA
moieties [1,2].
Metabolites 2018, 8, 37; doi:10.3390/metabo8020037
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Metabolites 2018, 8, 37
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The CA inhibitors (CAIs) belonging to the sulfonamide and sulfamate types have been
used clinically for several decades as diuretics [13 14], antiglaucoma agents [15], and anti-obesity
,
drugs [16,17]. More recently, a large number of studies showed that CA inhibition has profound
antitumor effects by inhibiting hypoxia-inducible isoforms hCA IX and XII, overexpressed in many
hypoxic tumors [18
of some CA isoforms in neuropathic pain [23
and coumarin [27 30] types demonstrating significant in vivo effects in animal models of these
–
22]. Furthermore, several proof-of-concept studies demonstrated the involvement
,
24] and arthritis [25 26], with the CAIs of sulfonamide
,
–
diseases. Thus, the field of drug design, synthesis, and in vivo investigations of various types
of CAIs is highly dynamic, with the action of a large number of interesting new chemotypes on
these widespread enzymes being constantly studied [27
between carbon dioxide (CO2) and bicarbonate with the formation of a proton, CAs are widespread
in organisms all over the phylogenetic tree as seven distinct genetic families: the -, -, -, -,
-, -, and -CAs [ 12 40 47]. CAs participate in crucial physiologic processes connected to pH
–39]. As they catalyze the interconversion
α
β
γ
δ
η
ξ
θ
3– , –
homeostasis, metabolism, transport of gases and ions, and secretion of electrolytes in virtually all
living beings [3–12,40–47].
Apart from the inhibition of human (h) or other vertebrate CA isoforms, the interest in inhibiting
such enzymes present in various pathogenic organisms (bacteria, fungi, protozoa, or worms) has
presented the possibility of designing anti-infective agents with a novel mechanism of action [40–51].
Thus, in this paper, we explored novel CAIs belonging to the sulfonamide class, incorporating
benzamide moieties similar to compounds reported earlier, but that were investigated for the inhibition
of isoforms involved in important diseases, such as glaucoma (hCA II), neuropathic pain (hCA VII),
or tumors (hCA IX), and ubiquitous off target isoform hCA I. Furthermore, we investigated whether
this chemotype shows inhibitory effects against β- and γ-class CAs from pathogenic bacteria (Vibrio
cholerae) or fungi (Malassezia globosa).
2. Results
2.1. Chemistry
The classical coupling of carboxylic acid
1 with amines, in the presence of carbodiimides (EDCI)
and hydroxybenzotriazole has been used for synthesis, as reported previously [1,2] (Scheme 1).
NH
SO2
2
O
Et N
R1
EDCI, HOAT,
DMF,
R2
3
+
HN
R2
N
R1
3-24h
dry
NH
SO2
2
O
OH
1
2
3
1
2
1
2
:
=
=H, R Et
3a R
=
dimethyl
3g R =H, R
D
-glutamate
,
=
1
2
1
2
n
=
-Pr
:
3b R
R
=
=H, R
L
3h R
methyl -leucinate
H,
H,
,
=
1
2
1
2
:
=
Propargyl
3c R
R
=
=H, R D
dimethyl
=
=H, R
methyl
3i R
,
3j R
,
-aspartate
-alaninate
-
1
3d R
R2
1
2
:
=
=
R2
CH
DL
O[(CH2 2)]2,
-
1
1
2
:
=
=
3e R
H
H
=
=H, R
ethyl 4-aminobutanoate
4C2 CH
3k R
,
[C2
2],
4
1
2
1
2
:
=
methyl
3f R =H, R
DL
-valinate
=
3l R =H, R
methyl
L
-phenylalaninate
,
Scheme 1. Synthesis of compounds 3a–l.
Compound
1
was condensed with compounds 3a
–e that possess primary or secondary amines as
well amino acid derivatives 3f
–
l
in the presence of EDCI and 1-hydroxy-7-azabenzotriazole (HOAT) to

Metabolites 2018, 8, 37
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obtain their corresponding amides (Scheme 1). By choosing variously substituted amines and amino
acids, incorporating both simple aliphatic and heterocyclic scaffolds (for the amine) and aliphatic and
aromatic amino acids, the physico-chemical properties and enzyme inhibitory properties of the new
compounds could be modulated. For example, the amino acid derivatives 3f, 3g, 3h, 3j, and 3l may
form sodium salts leading to water soluble CAIs.
2.2. Carbonic Anhydrase Inhibition
Sulfonamides 3a
I, II, VII, and IX, as well as three
bacterium Vibrio cholerae (VchCA ) and the fungus Malassezia globosa (MgCA), and the
same pathogenic bacterium, VchCA –enzymes recently cloned and characterized by our group as
potential anti-infective targets [52–59] (Table 1).
–
3l were tested as inhibitors of four hCAs involved in various pathologies, hCA
- and -CAs from pathogenic organisms: the -CAs from the
-CA from the
β
γ
β
β
γ
γ
Table 1. Inhibition data of human carbonic anhydrase (CA) isoforms hCA I, II, VII, IX, and pathogenic
bacteria and fungi - and -CAs with compounds 3a 3l in comparison with the standard sulfonamide
β
γ
–
inhibitor AAZ by a stopped flow carbon dioxide (CO2) hydrase assay [60].
KI (nM) a
Cpd
hCA I
hCA II
hCA VII
hCA IX
VchCAβ
MgCA
VchCAγ
3a
3b
3c
3d
3e
3f
3g
3h
3i
334
8.2
67.6
8.7
29.7
57.8
8.2
5.3
3.5
1.9
6.2
7.0
4.5
5.2
3.7
6.1
6.1
4.0
3.3
12.1
26.7
0.4
0.6
0.8
6.2
3.7
0.6
0.4
0.7
3.7
0.4
0.5
5.7
15.9
26.0
22.9
10.7
18.1
16.0
19.7
8.0
12.1
21.5
9.3
19.2
25.8
7082
7680
741
8587
749
8172
862
719
910
412
953
663
7669
3921
5781
5880
3985
5500
632
929
636
383
693
453
4458
503
891
744
271
756
409
473
5.6
763
75.7
85.3
5.3
5.6
250.0
6946
87.3
6695
517
3j
3k
3l
AAZ
451
74000
a
Mean from three different assay using a stopped flow technique. Errors were in the range of
reported values.
±
5% to 10% of the
3. Discussion
The following structure-activity relationship (SAR) were determined from the data of Table 1,
in which the standard sulfonamide inhibitor acetazolamide (AAZ) was also included for comparison.
The slow cytosolic isoform hCAI, involved in some ocular diseases (not glaucoma) [
inhibited by sulfonamides 3a reported here with KIs in the range of 5.3 to 334 nM. The ethyl- (3a
derivative was the weakest inhibitor, whereas 3c 3f 3i, and 3j showed medium potency inhibitory
action, with a KIs in the range of 57.8 to 85.3. These compounds incorporate propargyl, valyl, aspartyl,
and alanyl moieties. The remaining derivatives, 3b 3d 3e 3g 3h 3k, and 3l showed very effective
3–7], was
–l
)
,
,
,
,
,
,
,
hCA I inhibitory properties, with a KIs in the range of 5.3 to 29.7 nM, being CAIs an order of magnitude
better compared to acetazolamide (Table 1). Small changes in the scaffold (compare 3a and 3b) led to
dramatic changes in the hCA I inhibitory effects, with the propyl derivative 3b being 40.7 times more
effective an inhibitor compared with the ethyl derivative 3a.
All sulfonamides 3a–l reported here were excellent hCA II inhibitors, with a KIs in the range
of 1.9 to 7.0 nM, thus being more effective than AAZ (Table 1). With this highly effective inhibition
and small range in the variation of the KIs, the SAR is flat and the only conclusion is that all the
explored substitution patterns led to highly effective hCA II inhibitors. This is also the dominant

Metabolites 2018, 8, 37
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cytosolic isoform, involved in glaucoma, diuresis, respiration, and electrolyte secretion in a multitude
of tissues [3–12], meaning these results are highly significant.
The third cytosolic isoform investigated here, hCA VII, predominantly found in the brain
and involved in epileptogenesis and neuropathic pain [16
sulfonamides 3a , which showed a KIs in the range of 0.4 to 26.7 nM. Most of these compounds were
sub-nanomolar hCA VII inhibitors (e.g., 3b 3d, 3g 3i, 3k, 3l), being more effective by an order of
magnitude compared with the standard AAZ, whereas few of them showed the same potency as AAZ
3e 3f 3j) and only the ethyl derivative 3a was a less effective inhibitor compared to AAZ, with a KI
of 26.7 nM. Overall, the SAR is extremely simple, and except for the ethyl derivative mentioned above,
–24], was also effectively inhibited by
–
l
–
–
(
,
,
all the substitution patterns from derivatives 3b–3l indicated all compounds are highly effective hCA
VII inhibitors.
The tumor-associated, hypoxia-inducible isoform hCA IX was effectively inhibited by
sulfonamides 3a , with a KIs in the range of 8.0 to 26. 0 nMh. AAZ has an inhibition constant
of 25.8 nM against this isoform. The most effective inhibitors, 3h and 3k, with a KIs of 8.0–9.3 nM,
–
l
incorporated amino acyl moieties, but all substitution patterns present in compound
amino acid type, led to highly effective hCA IX inhibition.
3, of the amine or
Conversely, the
by these compounds, which showed activity in the micromolar range, with few exceptions (Table 1).
Thus, for VchCA , the KIs was in the range of 0.41 to 8.58 M; for MgCA, in the range of 87.3 nM to
7.67 M; and for VchCA , in the range of 0.27 to 4.45 M. Notably, 3j compounds, which incorporate
β- and γ-CAs from pathogenic organisms investigated here were poorly inhibited
β
µ
µ
γ
µ
the alanyl moiety, showed a good inhibitory effect against the Malassezia enzyme, one of the causative
agents of dandruff. Acetazolamide is a highly ineffective MgCA inhibitor, and most other sulfonamides
investigated here, although less effective than 3j, showed a better activity compared with the standard
sulfonamide CAI. Overall, β- and γ-CAs are less sensitive to inhibition with sulfonamides compared
with α-CAs [3–14].
4. Materials and Methods
4.1. Chemistry
Amines, 4-sulfamoyl-benzoic acid, buffers, solvents, and acetazolamide (AAZ) were commercially
available, obtained as highest purity reagents from Sigma-Aldrich/Merck, Milan, Italy. Nuclear
magnetic resonance (1H NMR, 13C NMR) spectra were recorded using a Bruker Avance III 400 MHz
spectrometer (Bruker, Billerica, MA, USA) in dimethyl sulfoxide (DMSO-d6I). Chemical shifts are
reported in parts per million (ppm) and the coupling constants (J) are expressed in Hertz (Hz). Splitting
patterns were designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; brs, broad singlet;
and dd, double of doubles. The assignment of exchangeable protons (OH and NH) was confirmed by
the addition of D2O. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel
F-254 plates. Flash chromatography purifications were performed on Merck Silica gel 60 (230–400 mesh
ASTM) as the stationary phase and MeOH/DCM were used as eluents.
4.1.1. General Procedure to Synthesize Compounds 3a–l
A solution of 4-carboxybenzene sulfonamide
3–5 mL) was treated with primary or secondary amines or amino acids 2a
1
(1.0 eq) in dry dimethylformamide (DMF,
(1.2 eq), then followed
–
l
by addition of N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDCI, 1.5 eq.),
1-hydroxy-7-azabenzotriazole (HOAT, 1.5 eq), and triethylamine (Et3N, 3 eq). The reaction continued
until the consumption of starting materials (TLC monitoring, 3–24 h) and quenched with water. The title
compounds were either obtained from filtration of the precipitates formed followed by washing with
water (3a
layers were washed with H2O (3
vacuum to provide a residue that was triturated from dichloromethane (3f–g, 3i–j).
–
3e,
3h, 3k
–
l) or extracted from ethyl acetate (EtOAc). In the latter, the combined organic
×
20 mL), dried over sodium sulfate, filtered, and concentrated in a

Metabolites 2018, 8, 37
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4.1.2. Characterization of Synthesized Compounds (3a–l)
N-Ethyl-4-Sulfamoylbenzamide (3a): 140 mg white solid, yield 83%; δH (400 MHz, DMSO-d6) 1.17 (3H,
t, J 7.2), 3.33 (2H, m), 7.51 (2H, s, exchange with D2O, SO2NH2), 7.93 (2H, d, J 8.4), 8.02 (2H, d, J 8.4),
8.68 (1H, t, J 7.2 exchange with D2O, NH); δC (100 MHz, DMSO-d6) 15.5, 35.1, 126.5, 128.6, 138.5, 147.0,
and 165.8; m/z (ESI positive) 229.0 [M + H]+.
N-Propyl-4-Sulfamoylbenzamide (3b): 120 mg white solid, yield 80%; δH (400 MHz, DMSO-d6) 0.93
(3H, t, J 7.2), 1.58 (2H, m), 3.27 (2H, q, J 7.2), 7.50 (2H, s, exchange with D2O, SO2NH2), 7.93 (2H, d, J
8.4), 8.02 (2H, d, J 8.4), 8.66 (1H, t, J 7.2, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 12.3, 23.2,
42.0, 126.5, 128.7, 138.5, 147.0, 166.0; m/z (ESI positive) 243.1 [M + H]+.
N-(Prop-2-Yn-1-Yl)-4-Sulfamoylbenzamide (3c): 120 mg yellow solid, yield 74%; δH (400 Mhz,
DMSO-d6) 3.18 (1H, T, J 2.5), 4.12 (2H, dd, J 5.5, 2.5), 7.52 (2H, s, exchange with D2O, SO2NH2),
7.94 (2H, d, J 8.8), 8.04 (2H, d, J 8.8), 9.16 (1H, t, J 5.5, exchange with D2O, NH);
δ (100 Mhz, DMSO-d )
c
6
29.5, 73.9, 81.9, 126.5, 128.8, 137.6, 147.3, 165.8; m/z (ESI Positive) 239.0 [M + H]+. Experimental data in
agreement with reported data [61].
4-(Morpholine-4-Carbonyl)Benzenesulfonamide (3d): 10 mg pale yellow solid; 9% yield; δH (400 MHz,
DMSO-d6) 3.65 (8H, m), 7.44 (2H, s, exchange with D2O, SO2NH2), 7.63 (2H, d, J 8.0), 7.92 (2H, d, J 8.0);
δC (100 MHz, DMSO-d6) 66.9, 66.9, 126.7, 128.5, 139.7, 145.8, 168.8; m/z (ESI positive) 271.1 [M + H]+.
4-(Piperidine-1-Carbonyl)Benzenesulfonamide (3e): 12 mg yellow solid, yield 18%; δH (400 MHz,
DMSO-d6) 1.50 (2H, m), 1.65 (4H, m), 3.25 (2H, m), 3.63 (2H, m), 7.48 (2H, s, exchange with D2O,
SO2NH2), 7.59 (2H, d, J 8.0), 7.91 (2H, d, J 8.0); δC (100 MHz, DMSO-d6) 26.1, 26.7, 48.8, 126.7, 128.0,
140.7, 145.4, 168.5; m/z (ESI positive) 269.1 [M + H]+.
Methyl (4-Sulfamoylbenzoyl)-DL-Valinate (3f): 12 mg pale yellow solid, yield 10%; δH (400 MHz,
DMSO-d6) 0.98 (3H, d, J 6.8), 1.02 (3H, d, J 6.8), 2.23 (1H, m), 3.70 (3H, s), 4.36 (1H, t, J 6.8), 7.53 (2H, s,
exchange with D2O, SO2NH2), 7.94 (2H, d, J 8.4), 8.05 (2H, d, J 8.4), 8.83 (1H, d, J 6.8, exchange with
D2O, NH); δC (100 MHz, DMSO-d6) 19.9, 20.0, 30.5, 52.6, 59.6, 126.4, 129.2, 137.7, 147.4, 167.0, 172.9;
m/z (ESI positive) 315.0 [M + H]+.
Dimethyl (4-Sulfamoylbenzoyl)-D-Glutamate (3g): 14 mg pale yellow solid, yield 16%; δH (400 Mhz,
DMSO-d6) 2.07 (2H, m), 2.16 (2H, m), 3.63 (3H, s), 3.70 (3H, s), 4.53 (1H, m), 7.52 (2H, s, exchange with
D2O, SO2NH2), 7.94 (2H, d, J 8.8), 8.05 (2H, d, J 8.8), 8.83 (1H, d, J 7.3, exchange with D2O, NH); δC
(100 Mhz, DMSO-d6) 26.6, 30.8, 52.3, 52.9, 53.0, 126.5, 129.0, 137.4, 147.5, 166.6, 172.9, 173.5; m/z (ESI
Positive) 359.1 [M + H]+.
Methyl (4-Sulfamoylbenzoyl)-L-Leucinate (3h): 37 mg white solid, yield 25%; δH (400 Mhz, DMSO-d6)
0.92 (3H, d, J 6.4), 0.97 (3H, d, J 6.4), 1.63 (1H, m), 1.70–1.86 (2H, m), 3.69 (3H, s), 4.56 (1H, m), 7.52
(2H, s, exchange with D2O, SO2NH2), 7.95 (2H, d, J 8.3), 8.06 (2H, d, J 8.3), 8.94 (1H, d, J 6.4, exchange
with D2O, NH); δC (100 Mhz, DMSO-d6) 22.1, 23.7, 25.3, 40.2, 51.9, 52.8, 126.5, 129.0, 137.5, 147.4, 166.5,
173.8; m/z (ESI Positive) 329.01 [M + H]+.
Dimethyl (4-Sulfamoylbenzoyl)-D-Aspartate (3i): 35 mg yellow solid, yield 50%; δH (400 Mhz,
DMSO-d6) 2.86–3.04 (2H, m), 3.67 (3H, s), 3.70 (3H, s), 4.89 (1H, m), 7.52 (2H, s, exchange with
D2O, SO2NH2), 7.96 (2H, d, J 8.7), 8.03 (2H, d, J 8.7), 9.13 (1H, d, J 7.6, exchange with D2O, NH); δC
(100 Mhz, DMSO-d6) 36.2, 50.2, 52.6, 53.2, 126.6, 129.0, 137.2, 147.6, 166.1, 171.3, 171.9; m/z (ESI Positive)
345.0 [M + H]+.
Methyl (4-Sulfamoylbenzoyl)-DL-Alaninate (3j): 14 mg white solid, yield 13%; δH (400 Mhz, DMSO-d6)
1.46 (3H, d, J 7.3), 3,69 (3H, s), 4.54 (1H, m), 7.51 (2H, s, exchange with D2O, SO2NH2), 7.96 (2H, d,
J 8.4), 8.06 (2H, d, J 8.4), 9.01 (1H, d, J 7.3, exchange with D2O, NH); δC (100 Mhz, DMSO-d6) 17.6, 49.3,
52.8, 126.5, 129.0, 137.4, 147.4, 166.2, 173.8; m/z (ESI Positive) 287.0 [M + H]+.

Metabolites 2018, 8, 37
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Ethyl 4-(4-Sulfamoylbenzamido)Butanoate (3k): 80 mg white solid, yield 58%; δH (400 Mhz, DMSO-d6)
1.20 (3H, t, J 7.2), 1.83 (2H, pent, J 6.8), 2.39 (2H, t, J 6.8), 3.31 (2H, m), 4.09 (2H, q, J 7.2), 7.47 (2H, s,
exchange with D2O, SO2NH2), 7.91 (2H, d, J 8.0), 8.01 (2H, d, J 8.0), 8.66 (1H, t, J 6.8, exchange with
D2O, NH); δC (100 Mhz, DMSO-d6) 15.0, 25.3, 31.9, 39.6, 60.6, 126.5, 128.7, 138.3, 147.1, 166.1, 173.5; m/z
(ESI Positive) 315.0 [M + H]+.
Methyl (4-Sulfamoylbenzoyl)-L-Phenylalaninate (3l): 130 mg white solid, yield 72%; δH (400 Mhz,
DMSO-d6) 3.10–3.25 (2H, m), 3.69 (3H, s), 4.70–4.76 (1H, m), 7.24 (1H, m), 7.32 (4H, m), 7.52 (2H, s,
exchange with D2O, SO2NH2), 7.93 (2H, d, J 8.4), 7.98 (2H, d, J 8.4), 9.08 (1H, d, J 7.9, exchange with
D2O, NH); δC (100 Mhz, DMSO-d6) 37.1, 52.9, 55.2, 126.5, 127.4, 128.9, 129.2, 130.0, 137.4, 138.4, 147.4,
166.3, 172.8; m/z (ESI Positive) 363.0 [M + H]+.
4.2. CA Enzyme Inhibition Assay
An Sx.18Mv-R Applied Photophysics (Oxford, U.K.) stopped-flow instrument was used to
assay he catalytic activity of various CA isozymes for CO2 hydration reaction [60]. Phenol red,
at a concentration of 0.2 mM, was used as an indicator, working at the absorbance maximum of 557 nm,
with 10 mM Hepes (pH 7.5, for α-CAs) or TRIS (pH 8.3, for β- and γ-CAs) as buffers, 0.1 M sodium
sulfate (Na2SO4) (for maintaining constant ionic strength), following the CA-catalyzed CO2 hydration
◦
reaction for a period of 10 s at 25 C. The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces
of the initial 5–10% of the reaction were used for determining the initial velocity. The uncatalyzed rates
were determined in the same manner and subtracted from the total observed rates. Stock solutions of
inhibitors (10 mM) were prepared in distilled-deionized water. Dilutions up to 1 nM were performed
thereafter with the assay buffer. Enzyme and inhibitor solutions were pre-incubated together for 15 min
(standard assay at room temperature) prior to assay, to allow for the formation of the enzyme–inhibitor
complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM
3 and the Cheng-Prusoff equation, as reported earlier [62
produced as reported earlier by our groups [52–76].
–75]. All CAs were recombinant proteins
5. Conclusions
We report a series of benzamides incorporating 4-sulfamoyl moieties, which were obtained
by reacting 4-sulfamoyl benzoic acid with primary and secondary amines and amino acids. These
sulfonamides were investigated as inhibitors of several enzymes, including the human (h) isoforms
hCA II, VII, and IX, involved in severe pathologies, such as glaucoma, epilepsy, neuropathic pain
and cancer; and β- and γ-class CAs from pathogenic bacteria and fungi. hCA II, VII, and IX were
inhibited in the low nanomolar or subnanomolar ranges by all investigated sulfonamides, whereas
hCA I was slightly less sensitive to inhibition (KIs of 5.3–334 nM). The Vibrio cholerae and Malassezia
globosa CAs were generally inhibited in the micromolar range by the sulfonamides reported in the
paper. The benzamide-4-sulfonamides constitute a promising class of highly effective CA inhibitors.
Further investigations will focus on extending the series of sulfanilamide possessing aliphatic tails
with carbamide linkers, such as cyclic and aliphatic and aromatic, to investigate and obtain isoform
selective inhibitors for their profiling and possible in vivo applications.
Author Contributions: M.A. synthesized and characterized the CAIs, A.A. performed the stopped-flow analysis.
M.B. supervised to study and edited the manuscript. C.T.S. wrote and edited the manuscript. All authors read
and approved the final paper
Funding: This research received no external funding
Conflicts of Interest: The authors declare no competing financial interest.

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