Design, synthesis, and biological evaluation of 1,2,3-triazole-linked triazino[5,6-b]indole-benzene sulfonamide conjugates as potent carbonic anhydrase I, II, IX, and XIII inhibitors

Article abstract of DOI:10.3390/metabo10050200

A series of 1,2,3-triazole-linked triazino[5,6-b]indole-benzene sulfonamide hybrids (6a– 6o) was synthesized and evaluated for carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity against the human (h) isoforms hCA I, II, XIII (cytosolic isoforms), and hCA IX (transmembrane tumor-associated isoform). The results revealed that the compounds 6a–6o exhibited K_i values in the low to medium nanomolar range against hCA II and hCA IX (K_is ranging from 7.7 nM to 41.3 nM) and higher K_i values against hCA I and hCA XIII. Compound 6i showed potent inhibition of hCA II (K_i = 7.7nM), being more effective compared to the standard inhibitor acetazolamide (AAZ) (K_i = 12.1 nM). Compounds 6b and 6d showed moderate activity against hCA XIII (K_i= 69.8 and 65.8 nM). Hence, compound 6i could be consider as potential lead candidate for the design of potent and selective hCA II inhibitors.

Full text of DOI:10.3390/metabo10050200

Article
Design, Synthesis, and Biological Evaluation of 1,2,3-
Triazole-Linked Triazino[5,6-B]Indole-Benzene
Sulfonamide Conjugates as Potent Carbonic
Anhydrase I, II, IX, and XIII Inhibitors
Krishna Kartheek Chinchilli 1, Andrea Angeli 2, Pavitra S. Thacker 1, Laxman Naik Korra 1,
Rashmita Biswas 1, Mohammed Arifuddin 1,3,* and Claudiu T. Supuran 2,*
1
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research
(NIPER), Balanagar, Hyderabad 500037, India; krishnakrthk22@gmail.com (K.K.C.);
thackerp42@yahoo.com (P.S.T.); laxmanaik827@gmail.com (L.N.K.); rashmitabiswas06@gmail.com (R.B.)
2
Neurofarba Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di
Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy; andrea.angeli@unifi.it
3
Department of Chemistry, Anwarul Uloom College, 11-3-918, New Malleypally,
Hyderabad-500001, T. S., India
* Correspondence: arifabib@gmail.com (M.A.); claudiu.supuran@unifi.it (C.T.S.)
Received: 5 April 2020; Accepted: 12 May 2020; Published: 15 May 2020
Abstract: A series of 1,2,3-triazole-linked triazino[5,6-b]indole-benzene sulfonamide hybrids (6a–
6o) was synthesized and evaluated for carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity
against the human (h) isoforms hCA I, II, XIII (cytosolic isoforms), and hCA IX (transmembrane
tumor-associated isoform). The results revealed that the compounds 6a–6o exhibited K
the low to medium nanomolar range against hCA II and hCA IX (K s ranging from 7.7 nM to 41.3
nM) and higher K values against hCA I and hCA XIII. Compound 6i showed potent inhibition of
hCA II (K = 7.7nM), being more effective compared to the standard inhibitor acetazolamide (AAZ)
(K = 12.1 nM). Compounds 6b and 6d showed moderate activity against hCA XIII (K = 69.8 and 65.8
i
values in
i
i
i
i
i
nM). Hence, compound 6i could be consider as potential lead candidate for the design of potent and
selective hCA II inhibitors.
Keywords: 1,2,3-triazole; triazino[5,6-b]indole-benzene sulfonamide; carbonic anhydrase inhibitors
1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are omnipresent metalloenzymes that they play a pivotal
catalytic role in the hydration of carbon dioxide to bicarbonate and protons by means of a ping pong
mechanism, which is a slow process under non-catalytic conditions [1–4].
ꢅ
ꢀꢁꢂ + ꢃꢂꢁ ⇔ ꢃꢀꢁꢄ + ꢃꢆ
These enzymes are encoded by eight genetically unrelated gene families, namely, α, β, γ, δ, ζ, η,
θ, and the recently reported ι class [5]. Among these, the α family is predominantly present in
mammals, and 16 isoforms have been reported with different catalytic activity, subcellular
localization, and tissue distribution [1–4]. There are five cytosolic forms (CA I, CA II, CA III, CA VII,
and CA XIII), five membrane-bound isozymes (CA IV, CA IX, CA XII, CA XIV, and CA XV), two
mitochondrial forms (CA VA and CA VB), and a secreted isozyme (CA VI) [1–4]. These isoforms are
implicated in different diseases, as shown in Table 1. Therefore, selective inhibition of a particular
isoform redresses the particular disease in which it plays a major role.
Metabolites 2020, 10, 200; doi:10.3390/metabo10050200
www.mdpi.com/journal/metabolites

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Table 1. Carbonic anhydrase (CA) isoforms and associated diseases.
Disease
Glaucoma
Isoform Target
CA II, CA IV, CA XII
CA IX, CA XII
CA VII
Cancer
Epilepsy
Antineuropathic pain
Obesity
CA VII
CA VA
To date, the sulfonamide group is considered as main zinc-binding group for the design of
carbonic anhydrase inhibitors. Sulfonamides and their bio-isosteres such as sulfamides / sulfamates
are known to elicit potent carbonic anhydrase inhibition and hence they are present in drugs, which
are prescribed for the treatment of glaucoma, epilepsy, obesity, and cancer. The diuretic drugs mainly
target CA II, CA IV, CA XII, and CA XIV [6,7], the anti-glaucoma drugs target CA II, CA IV, and CA
XII [8,9], while the anti-epileptics target CA VII and CAXIV [10–12]. CA IX and CA XII specifically
expressed in tumor cells, and their inhibition results in anti-metastatic effects [13–15]. However, the
main drawback of all these drugs is the lack of selectivity, which results in serious side effects.
Therefore, there is an urgent need to design and develop selective isoform inhibitors. The tail
approach has been very successful in addressing this issue, and many novel scaffolds have been
developed [16,17]. In this approach, the attached tails bind to the active site cavity, preferably the
middle and the rim part, which shows variation in different CA isoforms. Some clinically/pre-
clinically used sulfonamides are illustrated in the Figure 1:
Figure 1. Structures of some clinically used sulfonamides.
Owing to the development of novel carbonic anhydrase inhibitors with better isoform
selectivity, our group designed some novel hybrids in which the triazino[5,6-b]indole tail was
conjugated to benzene sulfonamide via a 1,2,3-triazole linker. Triazino[5,6-b]indole is a flexible tail
with diverse pharmacological activities, like anti-fungal/anti-bacterial [18], anti-diabetic [19], anti-
depressant [20], anti-hypertensive [21], anti-inflammatory [22], and anti-hypoxic activities [23].
The design of this new series of compounds was based on the tail approach via the fusion of
indole and 1,2,4-triazine, which were reported for high interactions with carbonic anhydrase (Figure
2) [24–28]. The present design mainly is mainly involved two strategies. The first one was to fuse the
two CA-binding scaffolds i.e., Indole and 1,2,4-triazine in order to develop a flexible tail with better
interactions in the enzymatic site and the second one was to incorporate different N-alkyl substituents
in the indole tail in a systematic fashion to define optimal length (methyl, ethyl, propyl), bulkiness
(isopropyl) and un-saturation (allyl), which would confer the best CA inhibitory activity. It is

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reported in the literature that 1,2,3-triazole is an efficient linker, useful in the design of potent CA
inhibitors, as it is an amide bioisostere and maintains high stability under basic as well as acidic
hydrolysis conditions. It also has high dipole moment and capability of H bonding in vivo. Due to its
aromatic character, it shows some π-stacking interactions with relevant amino acid residues [29].
Figure 2. Design of target molecules by a molecular hybridization approach for hCA: human carbonic
anhydrase
2. Results and Discussion
2.1. Synthesis of the Target Molecules
The current design of experiment (DOE) was based on the molecular hybridization approach.
We synthesized molecular hybrids of a bulky triazino[5,6-b]indole, used as a tail, conjugated to
benzene sulfonamide through a flexible 1,2,3-triazole linker.
The synthesis of 1,2,3-triazole-linked triazino[5,6-b]indole-benzene sulfonamide hybrids (6a–6o)
was performed according to the general synthetic scheme illustrated in Scheme 1. The intermediate
compounds (4a–o) were synthesized according to previously reported methods [30,31]. The N-
alkylated isatins (3a–o) were synthesized from the simple five-substituted isatins (1a–c) by
nucleophilic substitution of different alkyl halides (2a–e). The 3a-o were condensed with
thiosemicarbazide in aqueous 1,4-dioxane under reflux conditions by using cesium carbonate as a
base followed by propargylation to generate intermediates (5a–o.) Finally the 5a-o were subjected to
click reaction with 4-azido benzene sulfonamide to generate 1,2,3-triazole-linked triazino[5,6-
b]indole-benzene sulfonamide hybrids (6a–o).

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Scheme 1. Synthesis of target 1,2,3-triazole-linked triazino[5,6-b]indole-benzene sulfonamide hybrids
(6a–6o). Reagents and conditions: (i) K CO , KI (0.05 mole%), DMF, reflux, 4–6 h; yield: 72–75%; (ii)
thiosemicarbazide, Cs CO , 1,4-dioxane, reflux, overnight; yield: 68–70%; (iii) propargyl bromide,
CO , DMF, rt, overnight; yield: 86–90%; (iv) CuSO .5H O, sodium ascorbate, tBuOH:H O (1:1), 60
2
3
2
3
K2
3
4
2
2
°C, overnight; yield: 65–70%.
2.2. Carbonic Anhydrase Inhibition
The newly synthesized 1,2,3-triazole linked triazino[5,6-b]indole-benzene sulfonamide hybrids
(6a–6o) were evaluated for their carbonic anhydrase inhibitory activity against a panel of carbonic
anhydrases, i.e., hCA I, hCA II, hCA IX, and hCA XIII, by the stopped-flow CO
method. Highly purified CA isoforms were employed, for which the kinetic parameters for the
physiologic reaction (CO hydration) were measured (see the Experimental section for details),
monitoring the color change produced by the formation of H+ ions (and bicarbonate). For all the pure
enzymes, the kinetic parameters (kcat and kcat/K ) are measured and these values are given in the
2
hydrase assay
2
M
Table 2. These activities were highly inhibited by the clinically used sulfonamide inhibitor
acetazolamide (AAZ), as shown in Table 2. It was observe that all these enzymes are highly efficient
catalysts with kcat/K
> 107 M−1x s−1
M
Table 2. Kinetic parameters of the pure CA isoforms employed in this work and inhibition constants
for acetazolamide (AAZ), a standard sulfonamide drug.
K
(s−1)
cat
k
cat/K
M
K
I
(Acetazolamide)
Organisms CA Class Acronym
(M−1 × s−1)
(nM)
250
ꢇ
ꢇ
ꢇ
ꢇ
hCA I
2.0 × 105 5.0 × 107
1.4 × 106 1.5 × 108
hCA IX a 3.8 × 105 5.5 × 107
hCA_XIII 1.5 × 105 1.1 × 107
a Catalytic domain.
Homo sapiens
hCA II
12.1
25.8
17.0

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The following structure-activity relationship can be inferred from the inhibition data of
compounds 6a–o (Table 3).
I
The cytosolic hCA II isoform was strongly inhibited by all the synthesized compounds 6a–o, in
a low to medium nanomolar range (K s = 7.7 nM to 0.2527 µM). The best activity against hCA II
was shown by compound 6i (K = 7.7 nM), possessing a fluoro group attached at the 5th position
of the indole ring and an isopropyl group anchored to the nitrogen of indole. It was almost
twofold more active than the standard AAZ (K = 12.1 nM). Compounds 6d–6g, were found to
have potent activity at the nanomolar concentration against hCA II, with K ranging from 20.9 to
i
i
i
i
63.9 nM. Compounds 6k–6o, containing a chloro group at the 5th position of indole, showed
lower activity in the range of 61.7 to 252.7 nM, compared to compounds containing a fluoro
group and unsubstituted indole.
II The transmembrane hCA IX isoform, which is expressed exclusively in tumors, was also
strongly inhibited by the synthesized compounds in the medium nanomolar range (K s = 34.9
nM to 0.3246 µM). Compounds 6d, 6e, 6f, and 6i showed equipotent nanomolar activity with
AAZ, with K s ranging from 34.9 nM to 41.3 nM. Among these compounds, 6i showed the best
activity (K = 34.9 nM) against hCA IX isoform.
III The cytosolic hCA I and hCA XIII isoforms were inhibited by all synthesized compounds in the
high nanomolar range (K s > 500 nM). However, compounds 6b and 6d showed moderate
activity with K s of 69.8 nM and 65.8 nM respectively against hCA XIII isoform.
i
i
i
i
i
From the above structure–activity relationship, it was found that compound 6i was the most
potent compound with a K values of 7.7 nM against hCAII and 34.9 nM against hCA IX.
i
Table 3. Inhibition of hCA isoforms I, II, IX, and XIII with compounds 6a–o and AAZ as a standard
inhibitor
K
I
(nM)
hCA hCA hCA hCA
II IX XIII
Compound
Structure
I
6a
910.1 65.5 285.6 77.8
642.2 72.0 172.5 69.8
3960 88.7 219.4 364.8
6b
6c
6d
314.7 20.9
37.8
65.8

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6e
535.8 41.7
41.1 626.7
6f
6g
6h
766.2 59.6
41.3 834.8
698.5 63.9 193.1 675.0
764.2 682.7 118.6 1815
6i
6j
379.2
7.7
34.9 736.2
4592 73.7 401.7 793.6
5140 252.7 330.7 867.0
2837 184.6 150.4 3980
6k
6l

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6m
6513 61.7 204.5 823.8
6n
6o
571.9 179.2 320.8 91.3
694.4 97.1 324.6 2300
AAZ
250.0 12.1
25.8
17.0
3. Conclusion
In conclusion, we report here the synthesis of a series of 1,2,3-triazole-linked triazino[5,6-
b]indole-benzene sulfonamide hybrids (6a–6o). The structures of these compounds where confirmed
by different spectral and elemental analyses methods (Supplemental Data S1). The Biological
evaluation of sulfonamides was performed against hCA I, hCA II, hCA IX, and hCA XIII. All
compounds showed low to moderate inhibitory activity against hCA II and hCA IX isoforms, at
concentrations in the range between 7.7 nM and 0.3246 µM. Compound 6i emerged as a potent hCA
II and hCA IX inhibitor (K
i
= 7.7 nM against hCA II and 34.9 nM against hCA IX). The compounds 6b
of 69.8 nM and 65.8 nM,
and 6d was showed activity at medium nanomolar concentrations, with K
i
respectively, against hCA XIII isoform. Thus, the compound 6i can be emerged as a novel potential
lead compound to develop selective carbonic anhydrase inhibitors against the hCA II isoform.
4. Experimental Section
4.1. General Experimental Conditions
All the chemicals and solvents utilized as obtained from the suppliers. Wherever necessary,
anhydrous solvents are used. Thin-layer chromatography analysis (TLC), was carried-out by utilizing
Merck silica gel 60 F254 aluminum plates. A Stuart Digital Melting point Apparatus (SMP 30) was used
1
to determining the melting point of the compounds, which were uncorrected. The H and 13C NMR
spectra were recorded on Bruker Avance 500 MHz and 125 MHz respectively, with DMSO-d as the
6
solvent. The chemical shift values were calculated in ppm using TMS as the standard reference. The
HRMS were recorded on Agilent QTOF mass spectrometer 6540 series and were performed using ESI
techniques at 70 eV. All the newly synthesized analogs were evaluated in vitro for their inhibitory
activity against a panel of recombinant CA isoforms, i.e., hCA I, hCA II, hCA IX, and hCA XIII,
obtained inhouse, using the stopped-flow CO hydrase assay.
2
4.1.1. Synthesis of N-Alkylated Isatins (3a–o)
To a stirred solution of isatin (0.5 g, 0.00398 mole) in DMF (10 mL) we added potassium
carbonate (0.939 g, 0.006796 mole) and potassium iodide (0.05 mole%), and the resulting solution was
stirred for about 30 minutes. After the specified time interval, the respective alkyl halides (2a–e) was

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added and the resulting solution was allowed to reflux. The progress of the reaction was monitored
by using TLC. Upon the completion of the reaction as assessed by TLC, the reaction mixture was
poured into ice water, and the precipitated solid was collected, washed with water, and recrystallized
from ethanol to yield compounds (3a–o) (yield 72–75%) [31,32].
4.1.2. Synthesis of N-Alkylated Triazino[5,6-B]Indolethioether Derivatives (4a–o)
To a stirred solution of N-alkylated isatin (0.450 g, 0.002378 mol) in 40% aqueous 1,4-dioxane (5
mL), thiosemicarbazide (0.260 g, 0.00285 mol) and Cs
2
CO (0.720 g, 0.00285 mol) were added. The
3
resulting solution was refluxed overnight. Upon completion of the reaction (as determined by TLC),
the reaction mixture was cooled to rt. The solid byproducts were filtered off and the filtrate was
acidified with conc. HCl to pH 1–3. The obtained solids were collected washed with water and dried
to give yellow-colored solids which were used without any further purification (yield 68–70%)
[31,32].
4.1.3. Synthesis of N-Alkyl-3-Prop-2-Yn-1-Ylthio)-5h-[1,2,4]Triazino[5,6b]Indole Derivatives (5a–o)
To a stirred solution of compounds 4a–o (0.140 g, 0.00607 mole) in DMF (3 mL), K
2
CO (0.101 g,
3
0.000729 mol) and propargyl bromide (0.087 g, 0.000729 mol) were added. The resulting reaction
mixture was stirred at rt overnight. Upon completion of the reaction (monitored by TLC), the reaction
mixture was poured in ice-cold water, and the formed solid was collected, washed with water, and
dried to give brown-colored solids that were used without any further purification (yield 86–90%).
4.1.4. Synthesis of 4-(4-(N-Alkyl-5h-[1,2,4]Triazino[5,6b]Indol-3-Yl)Thio)Methyl)- 1h-1,2,3-Triazol-1-
Yl)Benzenesulfonamide Derivatives (6a–o)
The compounds 5a–o (0.04 g, 0.0001 mol) and 4-azido benzene sulfonamide (0.024 g, 0.000 mol)
were suspended in 2 mL of a 1:1 water/tert-butanol mixture. Sodium ascorbate (0.048 g, 0.0002 mol)
was added, followed by copper (II) sulfate pentahydrate (0.031 g, 0.0001 mol). The heterogeneous
mixture was stirred vigorously overnight at which point it cleared and TLC analysis indicated
complete consumption of the starting materials. The reaction mixture were diluted with water and
cooled in ice to obtained the brown precipitate, which were collected by filtration. After washing with
cold water the precipitate were dried under vacuum to afford a pure product as a brown amorphous
solid (6a–o).
4-(4-(((5-methyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6a) Yield: 57%; Color: Brown solid; mp: 235–240 °C; 1H NMR (500 MHz, DMSO-d
) δ 8.87
6
(s, 1H), 8.34 (s, 1H), 8.10 (s, 2H), 7.99 (s, 2H), 7.80 (s, 2H), 7.51 (s, 3H), 4.77 (s, 2H), 3.86 (s, 3H);13C
NMR (125 MHz, DMSO) δ 167.1, 159.9, 158.0, 147.2, 145.7, 144.2, 141.3, 139.0, 138.5, 127.9, 122.5, 120.8,
113.2, 108.1, 107.9, 28.1, 25.29. HR-MS (ESI-QTOF): m/z calculated for [M + H]+ C19
H
16N
8O
2
S ; 453.0916;
2
found 453.0965.
4-(4-(((5-ethyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6b) Yield: 59%; Color: Brown solid; mp: 202–204 °C; 1H NMR (500 MHz, DMSO-d
6) δ8.85
(s, 10H), 8.38 (dd, J = 17.8, 4.8 Hz, 11H), 8.10 (d, J = 8.6 Hz, 21H), 7.99 (d, J = 8.6 Hz, 20H), 7.85 (d, J =
8.2 Hz, 8H), 7.83 – 7.75 (m, 12H), 7.50 (s, 20H), 4.75 (s, 19H), 4.44 (q, J = 7.0 Hz, 21H), 1.35 (t, J = 7.2 Hz,
28H); 13C NMR (125 MHz, DMSO-d
127.91, 123.28, 122.37, 122.06, 120.72, 117.94, 111.68, 36.45, 25.31, 13.73; HR-MS (ESI-QTOF): m/z
calculated for [M + H]+ C20
; 467.1072; found 467.1120.
6
) δ 166.58, 146.11, 145.83, 144.23, 141.55, 141.02, 139.02, 131.43,
H
18
N
8O2S2
4-(4-(((5-propyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
1
sulfonamide (6c) Yield: 61%; Color: Brown solid; mp: 261–263 °C; H NMR (500 MHz, DMSO) δ 8.62
(s, 1H), 8.13 (s, 1H), 7.81 (d, J = 56.5 Hz, 5H), 7.57 (d, J = 35.0 Hz, 3H), 7.27 (s, 3H), 4.51 (s, 2H), 4.12 (s,
2H), 1.57 (s, 3H); 13C NMR (125 MHz, DMSO) δ 166.7, 146.6, 144.2, 141.4, 139.0, 131.4, 127.9, 123.2,
122.3, 121.9, 120.7, 117.9, 111.8, 43.0, 25.35, 21.62, 11.6.HR-MS (ESI-QTOF): m/z calculated for [M + H]+
C21H
20N
8O
2
S ; 481.1229; found 481.1268.
2

Metabolites 2020, 10, 200
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4-(4-(((5-isopropyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6d) Yield: 53%; Color: Brown solid; mp: 213–215 °C; 1H NMR (500 MHz, DMSO-d
6
) δ 8.84
(s, 1H), 8.19 (d, J = 7.5 Hz, 1H), 8.09 (d, J = 8.4 Hz, 2H), 8.04 – 7.93 (m, 4H), 7.62 (t, J = 8.1 Hz, 1H), 7.49
(s, 2H), 5.21 – 5.13 (m, 1H), 4.73 (s, 2H), 1.61 (d, J = 6.7 Hz, 6H). 13C NMR (125 MHz, DMSO) δ 166.2,
146.2, 145.9, 144.2, 141.5, 140.6, 139.0, 131.3, 127.9, 123.1, 122.3, 122.0, 120.7, 118.2, 112.6, 46.7, 25.3, 20.3.
HR-MS (ESI-QTOF): m/z calculated for [M + H]+ C21
H
20
N
8
O
2
2
S ; 481.1229; found 481.1294.
4-(4-(((5-allyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6e) Yield: 61%; Color: Brown solid; mp: 230–232 °C; 1H NMR (500 MHz, DMSO-d
) δ8.84
6
(s, 1H), 8.39 (s, 1H), 8.05 (d, J = 45.9 Hz, 5H), 7.78 (s, 2H), 7.52 (s, 2H), 6.02 (s, 1H), 5.08 (s, 4H), 4.77 (s,
2H); 13C NMR (125 MHz, DMSO) δ 166.4, 147.0, 146.5, 144.3, 141.5, 141.3, 139.0, 131.4, 123.5, 122.5,
122.0, 120.8, 117.9, 112.0, 43.5, 25.3. HR-MS (ESI-QTOF): m/z calculated for [M + H]+ C21
H
10
N
8
O
2
2
S ;
479.1072; found 479.1087.
4-(4-(((8-fluoro-5-methyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6f) Yield: 59%; Color: Brown solid; mp: 244–246 °C; 1H NMR (500 MHz, DMSO-d
) δ8.86
(s, 1H), 8.20 – 8.16 (m, 1H), 8.10 (d, J = 8.6 Hz, 2H), 7.99 (d, J = 8.6 Hz, 2H), 7.84 (dd, J = 8.8, 3.9 Hz,
1H), 7.70 – 7.63 (m, 3H), 7.51 (s, 1H), 4.76 (s, 2H), 3.86 (s, 3H); 13C NMR (125 MHz, DMSO-d
) δ 167.15,
159.89, 147.16, 144.25, 139.02, 138.48, 127.90, 122.49, 120.78, 118.93, 118.73, 118.67, 118.59, 113.14,
6
6
107.91, 28.04, 25.29; HR-MS (ESI-QTOF): m/z calculated for [M + H]+ C19
H
15FN8O2
S2;471.0822; found
471.0824.
4-(4-(((8-fluoro-5-ethyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6g) Yield: 53%; Color: Brown solid; mp: 246–248 °C; 1H NMR (500 MHz, DMSO-d
) δ8.85
6
(s, 1H), 8.19 (dd, J = 8.1, 2.3 Hz, 1H), 8.10 (d, J = 8.7 Hz, 2H), 7.99 (d, J = 8.7 Hz, 2H), 7.91 (dd, J = 8.9,
4.0 Hz, 1H), 7.66 (td, J = 9.2, 2.4 Hz, 1H), 7.50 (s, 2H), 4.75 (s, 2H), 4.45 (q, J = 7.0 Hz, 2H), 1.34 (t, J = 7.1
Hz, 3H); 13C NMR (125 MHz, DMSO-d
122.39, 120.73, 118.97, 118.87, 118.77, 113.24, 108.09, 36.63, 25.33, 13.73; HR-MS (ESI-QTOF): m/z
calculated for [M + H]+ C20
17FN ; 485.0978; found 485.0979.
6
) δ 167.16, 159.85, 146.63, 145.75, 144.26, 139.03, 137.40, 127.91,
H
8O2S2
4-(4-(((8-fluoro-5-propyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6h) Yield: 63%; Color: Brown solid; mp: 240–242 °C; 1H NMR (500 MHz, DMSO) δ 8.84
(s, 1H), 8.18 (dd, J = 8.2, 2.5 Hz, 1H), 8.09 (d, J = 8.8 Hz, 2H), 7.98 (d, J = 8.8 Hz, 2H), 7.89 (dd, J = 9.0,
4.0 Hz, 1H), 7.64 (td, J = 9.2, 2.6 Hz, 1H), 7.50 (s, 2H), 4.74 (s, 2H), 4.35 (t, J = 6.9 Hz, 2H), 1.83 – 1.74
(m, 2H), 0.78 (t, J = 7.4 Hz, 3H); 13C NMR (125 MHz, DMSO) δ 167.3, 159.8, 157.9, 147.1, 145.8, 144.3,
141.1, 139.0, 137.8, 127.9, 122.3, 120.7, 113.4, 108.2, 107.9, 43.2, 25.4, 21.6, 11.5. HR-MS (ESI-QTOF): m/z
calculated for [M + H]+ C21
H
19 FN
8
O2
S 499.1135; found 499.1153.
2
4-(4-(((8-fluoro-5-isopropyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-
yl)benzene sulfonamide (6i) Yield: 55%; Color: Brown solid; mp: 235–237 °C; 1H NMR (500 MHz, DMSO-
d ) δ8.84 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.10 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.3 Hz, 3H), 7.63 (s, 1H),
6
7.50 (s, 2H), 5.17 (s, 1H), 4.73 (s, 2H), 1.61 (d, J = 6.1 Hz, 6H); 13C NMR (125 MHz, DMSO) δ 166.8,
159.7, 157.8, 146.7, 145.8, 144.2, 141.1, 139.0, 137.0, 127.9, 122.3, 120.7, 119.2, 118.8, 46.9, 25.3, 20.3. HR-
MS (ESI-QTOF): m/z calculated for [M + H]+ C21
4-(4-(((8-fluoro-5-allyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6j) Yield: 51%; Color: Brown solid; mp: 245–247 °C; 1H NMR (500 MHz, DMSO-d
) δ 8.81
H
19 FN8O2S
2499.1135; found 499.1174.
6
(s, 1H), 8.21 (d, J = 6.7 Hz, 1H), 8.09 (d, J = 8.3 Hz, 2H), 7.99 (d, J = 8.3 Hz, 2H), 7.78 (d, J = 5.0 Hz, 1H),
7.65 (t, J = 8.2 Hz, 1H), 7.50 (s, 2H), 6.06-5.96 (m, 1H), 5.13 – 5.05 (m, 4H), 4.74 (s, 2H); 13C NMR (125
MHz, DMSO) δ 167.4, 159.9, 158.0, 146.9, 145.7, 144.3, 141.3, 139.0, 137.6, 132.1, 127.9, 122.4, 120.8,
119.0, 118.7, 113.6, 108.3, 43.8, 25.3. HR-MS (ESI-QTOF): m/z calculated for [M + H]+ C21
H
17FN8O2S2
497.0978; found 497.1027.
4-(4-(((8-chloro-5-methyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6k) Yield: 62%; Color: Brown solid; mp: 243–248 °C; 1H NMR (500 MHz, DMSO) δ 8.87
(s, 1H), 8.39 (s, 1H), 8.10 (d, J = 8.1 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.84 (d, J = 5.5 Hz, 1H), 7.51 (s, 1H),
4.77 (s, 1H), 3.86 (s, 1H);13C NMR (126 MHz, CDCl
143.71, 135.73, 132.74, 127.25, 126.31, 125.47, 124.14, 118.20, 109.43, 41.18, 29.99. HR-MS (ESI-QTOF):
m/z calculated for [M + H]+ C19
487.0526; found 487.0530.
3) δ 172.04, 151.12, 150.44, 148.97, 145.57, 144.27,
H
15ClN
8
O2S2

Metabolites 2020, 10, 200
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4-(4-(((8-chloro-5-ethyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6l) Yield: 57%; color: Brown solid; mp: 252–256 °C; 1H NMR (500 MHz, DMSO) δ 8.86 (s,
1H), 8.40 (s, 1H), 8.10 (d, J = 7.3 Hz, 1H), 7.99 (d, J = 7.0 Hz, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.82 (s, 1H),
7.51 (s, 1H), 4.75 (s, 1H), 4.44 (d, J = 5.7 Hz, 1H), 1.29 (d, J = 47.6 Hz, 1H); 13C NMR (126 MHz, CDCl
δ 172.04, 151.80, 150.30, 149.06, 145.56, 145.35, 143.81, 132.66, 132.51, 127.17, 126.07, 125.52, 123.87,
3
)
122.79, 118.21, 53.69, 32.67, 29.99.HR-MS (ESI-QTOF): m/z calculated for [M + H]+ C20
H
18ClN
8
O S
2 2
501.0682; found 501.0694.
4-(4-(((8-chloro-5-propyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6m) Yield: 63%; Color: Brown solid; mp: 257–262 °C; 1H NMR (500 MHz, DMSO) δ 8.85
(s, 1H), 8.39 (s, 1H), 8.10 (d, J = 7.1 Hz, 1H), 7.99 (d, J = 7.0 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.80 (d, J =
7.3 Hz, 1H), 7.51 (s, 1H), 4.74 (s, 1H), 4.35 (s, 1H), 1.78 (d, J = 6.1 Hz, 1H), 0.79 (s, 2H); 13C NMR (126
MHz, DMSO) δ 167.46, 146.95, 145.67, 144.25, 140.63, 139.99, 138.86, 131.10, 127.90, 127.75, 122.34,
121.44, 120.70, 119.24, 113.68, 43.18, 25.42, 21.48, 11.56.HR-MS (ESI-QTOF): m/z calculated for [M + H]+
C21H
20ClN
8
O2
S2 515.0839; found 515.0839.
4-(4-(((8-chloro-5-isopropyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-
1
yl)benzene sulfonamide (6n) Yield: 56%; Color: Brown solid; mp: 263–265 °C; H NMR (500 MHz,
DMSO) δ 8.85 (s, 1H), 8.40 (s, 1H), 8.10 (d, J = 8.6 Hz, 1H), 7.99 (d, J = 8.6 Hz, 1H), 7.95 (d, J = 8.9 Hz,
1H), 7.80 – 7.75 (m, 1H), 7.50 (s, 1H), 5.16 (dt, J = 13.6, 6.7 Hz, 1H), 4.73 (s, 1H), 1.61 (d, J = 6.8 Hz,
1H);13C NMR (126 MHz, DMSO) δ 166.92, 146.52, 145.82, 144.26, 140.54, 139.11, 139.01, 130.89, 127.92,
127.53, 122.36, 121.33, 120.73, 119.71, 114.31, 47.03, 30.95, 25.38, 20.33.HR-MS (ESI-QTOF): m/z
calculated for [M + H]+ C21
H
20ClN
8O2
S2 515.0839; found 515.0856.
4-(4-(((8-chloro-5-allyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene
sulfonamide (6o) Yield: 67%; Color: Brown solid; mp: 261–264 °C; 1H NMR (500 MHz, TFE) δ 8.81 (s,
1H), 8.40 (s, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.98 (d, J = 8.7 Hz, 1H), 7.81 – 7.75 (m, 1H), 7.50 (s, 1H), 5.99
(ddd, J = 22.0, 10.3, 5.0 Hz, 1H), 5.13 – 5.02 (m, 2H), 4.73 (s, 1H);13C NMR (126 MHz, DMSO) δ 167.54,
146.67, 145.70, 144.27, 140.68, 139.65, 138.99, 131.82, 131.04, 127.89, 122.51, 121.44, 120.76, 119.42,
117.79, 113.91, 43.48, 31.33, 25.06.HR-MS (ESI-QTOF): m/z calculated for [M + H]+ C21
H
17ClN
8
O2S2
513.0682; found 513.069.
4.2. CA Inhibition
An SX.18 V-R Applied Photophysics (Oxford, UK) stopped-flow instrument was used to assay
the catalytic inhibition of various CA enzymes [32]. Phenol Red (at a concentration of 0.2 mM) was
used as an indicator, working at maximum absorbance of 557 nm, with 10 mM Hepes (pH 7.4) buffer,
0.1 M Na
concentration), following the CA-catalyzed CO
CO solutions in water at 25 °C were used as a substrate. Stock solutions of the inhibitors were
2
SO
4
(for maintaining constant ionic strength; these anions are not inhibitory in the used
2
hydration reaction for a period of 5–10 s. Saturated
2
prepared at a concentration of 10 mM (in DMSO/Water 1:1, v/v), and dilutions up to 0.01 nM were
prepared using the assay buffer mentioned above. At least 7 different inhibitor concentrations were
used for measuring the inhibition constant. Inhibitor (I) and enzyme (E) solutions were pre-incubated
together for 10 min at room temperature prior to the assay, in order to allow for the formation of the
E–I complex. IC50-s values were calculated from the enzyme activity (as kcat, see Table 2) with respect
to the inhibitor concentration. Triplicate experiments were done for each inhibitor concentration, and
the values reported in this paper are the mean of such results. The inhibition constants were obtained
by non-linear least-square methods, using the Cheng–Prusoff equation, as reported earlier, and
represent the mean from at least three different determinations: K
I
= IC50/[(1 + [S]/K
M
)]. K values for
M
all enzymes were reported earlier (see Table 2) by us [26–31]; [S] is the CO
2
concentration at which
the experiments were performed. All CA isozymes used here were recombinant proteins obtained as
reported earlier by our group [33–38]
Supplementary Materials: The spectral data as supporting information are available online at
www.mdpi.com/2218-1989/10/5/200/s1; Data S1: 1H NMR spectra of 6a–o, 13C NMR spectra of 6a–o.
Author Contributions: The authors (K.K.C., P.S.T., L.N.K., R.B., M.A.) from NIPER-Hyderabad are involved in
the design and synthesis of the target molecules, whereas the authors (A.A., C.T.S.) from Università degli Studi

Metabolites 2020, 10, 200
11 of 12
di Firenze are involved in the biological evaluations. All authors have read and agreed to the published version
of the manuscript.
Funding: This research received no external funding.
Acknowledgments: KKC: PST, LNK, and RB are thankful to the Department of Pharmaceuticals, Ministry of
Chemicals and fertilizers, Govt. of India, New Delhi, for awarding NIPER Fellowships. The Italian Ministry for
University and Research, MIUR is also thanked for a grant to CTS (PRIN: rot. 2017XYBP2R).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Alterio, V.; Di Fiore, A.; Ambrosio, K.D.; Supuran, C.T.; de Simone, G. Multiple binding modes of inhibitors
to carbonic anhydrases: How to design specific drugs targeting 15 different isoforms? Chem. Rev. 2012, 112,
4421–4468.
2.
Supuran, C.T. Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators. Nat. Rev.
Drug Discov. 2008, 7, 168–181.
3.
4.
5.
Supuran, C.T. Structure and function of carbonic anhydrases. Biochem. J. 2016, 473, 2023–2032.
Supuran, C.T. Carbonic anhydrases and metabolism. Metabolites 2018, 8, 25.
Jensen, E.L.; Clement, R.; Kosta, A.; Maberly, S.C.; Gontero. B. A new wide spread sub class of carbonic
anhydrase in marine phytoplankton. ISME J. 2019, 13, 2094–2106.
6.
Lock, F.E.; McDonald, P.C.; Lou, Y.; Serrano, I.; Chafe, S.C.; Ostlund, C.; Aparicio, S.; Winum, J.Y.; Supuran,
C.T.; Dedhar. S. Targeting carbonic anhydrase IX depletes breast cancer stem cells within the hypoxic niche.
Oncogene 2013, 32, 5210–5219.
7.
8.
Bayram, E.; Senturk, M.; Kufrevioglu, O.I.; Supuran. C.T. In vitro inhibition of salicylic acid derivatives on
human cytosolic carbonic anhydrase isozymes I and II. Bioorg. Med. Chem. 2008, 16, 9101–9105.
Bozdag, M.; Pinard, M.; Carta, F.; Masini, E.; Scozzafava, A.; McKenna, R.; Supuran. C.T. A Class of 4-
Sulfamoylphenyl-ω-aminoalkyl Ethers with Effective Carbonic Anhydrase Inhibitory Action and
Antiglaucoma Effects. J. Med. Chem. 2014, 57, 9673–9686.
9.
Chegaev, K.; Lazzarato, L.; Tamboli, Y.; Boschi, D.; Blangetti, M.; Scozzafava, A.; Carta, F.; Masini, E.;
Fruttero, R.; Supuran, C.T.; Gasco. A. Furazan and furoxan sulfonamides are strong α-carbonic anhydrase
inhibitors and potential anti-glaucoma agents, Bioorg. Med. Chem. 2014, 22, 3913–3921.
10. De Luca, L.; Ferro, S.; Damiano, F.M.; Supuran, C.T.; Vullo, D.; Chimirri, A.; Gitto, R. Structure-based
screening for the discovery of new carbonic anhydrase XII inhibitors, Eur. J. Med. Chem. 2014, 71, 105–111.
11. Bruno, E.; Buemi, M.R.; De Luca, L.; Ferro, S.; Monforte, A.M.; Supuran, C.T.; Vullo, D.; De Sarro, G.; Russo,
E.; Gitto. R. In Vivo Evaluation of Selective Carbonic Anhydrase Inhibitors as Potential Anticonvulsant
Agents. ChemMedChem 2016, 11, 1812–1818.
12. Scozzafava, A.; Menabuoni, L.; Mincione, F.; Supuran. C.T. Carbonic anhydrase inhibitors: A general
approach for the preparation of water-soluble sulfonamides incorporating polyamino-polycarboxylate tails
and of their metal complexes possessing long-lasting, Topical intraocular pressure-lowering properties. J.
Med. Chem. 2002, 45, 1466–1476.
13. Tars. K.; Vullo, D.; Kazaks, A.; Leitans, J.; Lends, A.; Grandane, A.; Zalubovskis, A.; Scozzafava, A.;
Supuran. C.T. Sulfocoumarins (1,2-Benzoxathiine-2,2-dioxides): A Class of Potent and Isoform-Selective
Inhibitors of Tumor-Associated Carbonic Anhydrases. J. Med. Chem. 2013, 56, 293–300.
14. Maresca, A.; Scozzafava, A.; Supuran, C.T. 7,8-Disubstituted-but not 6,7-disubstituted coumarins
selectively inhibit the transmembrane, tumor associated carbonic anhydrase IX and XII over the cytosolic
ones I and II in the low nanomolar/subnanomolar range. Bioorg. Med. Chem. Lett. 2010, 20, 7255–7258.
15. Innocenti, A.; Sarıkaya, S.B.O.; Gulcin, I.; Supuran. C.T. Carbonic anhydrase inhibitors, Inhibition of
mammalian isoforms I-XIV with a series of natural product polyphenols and phenolic acids. Bioorg. Med.
Chem. 2010, 18, 2159–2164.
16. Pinard, M.A.; Mahon, B.; McKenna. R. Probing the surface of human carbonicanhydrase for clues towards
the design of isoform specific inhibitors. Biomed. Res. Int. 2015, 2015, 453543.
17. Aggarwal, M.; Kondeti, B.; McKenna. R. Insights towards sulfonamide drug specificity in alpha-carbonic
anhydrases. Bioorg. Med. Chem. 2013, 21, 1526–1533.
18. Kgokong, J.L.; Smith, P.P.; Matsabisa, G.M. 1,2,4-Triazino-[5,6b]indole derivatives: Effects of
trifluoromethyl group on in-vitro antimalarial activity. Bioorg. Med. Chem. 2005, 13, 2935–2942.
19. Rahim, F.; Ullah, K.; Ullah. H. Triazinoindole analogs as potent inhibitors of α-glucosidase: Synthesis,
biological evaluation and molecular docking studies. Bioorg. Med. Chem. 2015, 58, 81–87.

Metabolites 2020, 10, 200
12 of 12
20. Shelke, S.M.; Bhosale. S.H. Synthesis, antidepressant evaluation and QSAR studies of novel 2-(5H-
[1,2,4]triazino[5,6b]indol3-ylthio)-N-(substituted phenyl)acetamides. Bioorg. Med. Chem. Lett. 2010, 20,
4661–4664.
21. Monge, A.; Palop, J.A.; Ramierz, C.; Font, M.; Fernandez. A.E. New 5H-1,2,4-triazino[5,6b]indole and
aminoindole derivatives. Synthesis and studies as inhibitors of blood platelet aggregation, anti-
hypertensive agents and thromboxane synthetase inhibitors. Eur. J. Med. Chem. 1991, 26, 179–188.
22. Aswar, U.M.; Kalshetti, P.P.; Shelke, S.M.; Bhosale, S.H.; Bodhankar. S.L. Effect of newly synthesized 1,2,4-
triazino[5,6b]indole-3-thione derivatives on olfactory bulbectomy induced depression in rats. Asian. J. Trop.
Biomed. 2012, 2, 992–998.
23. Tomchin, A.B.; Uryupov, O.Y.; Zhukova, T.I.; Kuznetsova. T.A. Thiourea and thiosemicarbazide
derivatives: Structure, transformations, and pharmacological activity. Part III. Antihypoxic and anti-
inflammatory activity of 1,2,4-triazino[5,6b]indole derivatives. Pharma. Chem. J. 1997, 31, 632–637.
24. Demir-Yazici, K.; Bua, S.; Supuran. C.T. Indole-Based Hydrazones Containing A Sulfonamide Moiety as
Selective Inhibitors of Tumor-Associated Human Carbonic Anhydrase Isoforms IX and XII. Int. J. Mol. Sci.
2019, 20, 2354.
25. Peerzada, M.N.; Khan, P.; Ahmad, K. Synthesis, characterization and biological evaluation of tertiary
sulfonamide derivatives of pyridyl-indole based heteroaryl chalcone as potential carbonic anhydrase IX
inhibitors and anticancer agents. Eur. J. Med. Chem. 2018, 155, 13–23.
26. Eldehna, W.M.; Nocentini, A.; Hassan, G.S.; Supuran. C.T. Tumor-associated carbonic anhydrase isoform
IX and XII inhibitory properties of certain isatin-bearing sulfonamides endowed with in-vitro antitumor
activity towards colon cancer. Bioorg. Med. Chem. 2018, 81, 425–432.
27. Mojzych, M.; Ceruso, M.; Supuran. C.T. New pyrazolo[4,3e][1.2.4]triazino sulfonamides as carbonic
anhydrase inhibitors. Bioorg. Med. Chem. Lett. 2015, 23, 3674–3680.
28. Matysiak, J.; Skrzypek, A.; Tarasiuk, P. QSAR study of pyrazolo[4,3e][1,2,4]triazino sulfonamide against
tumor-associated human carbonic anhydrase isoforms IX and XII. Comp. Boil. Chem. 2017, 71, 57–62.
29. Nocentini, A.; Ferraroni, M.; Carta, F.; Supuran, C.T. Benzene sulfonamides incorporating flexible Triazole
Moieties Are Highly Effective Carbonic anhydrase Inhibitors: Synthesis and Kinetic, Crystallographic,
Computational and Intraocular Pressure Lowering Investigations. J. Med. Chem. 2016, 59, 10692–10704.
30. Huy, X.N.; Green, K.D.; Gajadeera. C.S. Potent 1,2,4-Triazino[5,6b]indole-3-thioether inhibitors of
Kanamycin Resistance Enzyme Eis from Mycobacterium tuberculosis. ACS Infect. Dis. 2018, 4, 1030–10440.
31. Patel, D.V.; Patel, N.R.; Kanhed. A.M. Novel Multitarget Directed Triazinoindole Derivatives as Anti-
Alzheimer Agents. ACS. Chem. Neurosci. 2019, 10, 3635–3661.
32. Khalifah. R.G. The carbon dioxide hydration activity of carbonic anhydrase. I. Stopflowkinetic studies on
the native human isoenzymes B and C. J. Biol. Chem. 1971, 246, 2561–2573.
33. Supuran, C.T.; Barboiu, M.; Luca, C.; Pop, E.; Brewster, M.E.; Dinculescu, A. Carbonic anhydrase activators.
Part 14. Synthesis of mono- and bis- pyridinium salt derivatives of 2-amino-5-(2-aminoethyl)- and 2-amino-
5-(3-aminopropyl)- 1,3,4-thiadiazole, and their interaction with isozyme II. Eur. J. Med. Chem. 1996, 31, 597–
606.
34. Öztürk Sarıkaya, S.B.; Gülçin, I.; Supuran, C.T. Carbonic anhydrase inhibitors. Inhibition of human
erythrocyte isozymes I and II with a series of phenolic acids Chem. Biol. Drug Des. 2010, 75, 515–520.
35. Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Supuran, C.T. Dithiocarbamates: A new
class of carbonic anhydrase inhibitors. Crystallographic and kinetic investigations. Chem Commun. 2012, 48,
1868–1870.
36. Boztaş, M.; Çetinkaya, Y.; Topal, M.; Gülçin, İ.; Menzek, A.; Şahin, E.; Tanc, M.; Supuran, C.T. Synthesis
and carbonic anhydrase isoenzymes I, II, IX, and XII inhibitory effects of dimethoxybromophenol
derivatives incorporating cyclopropane moieties. J. Med. Chem. 2015, 58, 640–650.
37. De Simone, G.; Supuran, C.T. (In)organic anions as carbonic anhydrase inhibitors. J. Inorg. Biochem. 2012,
111, 117–129.
38. Innocenti, A.; Gülçin, I.; Scozzafava, A.; Supuran, C.T. Carbonic anhydrase inhibitors. Antioxidant
polyphenols effectively inhibit mammalian isoforms I–XV. Bioorg. Med. Chem. Lett. 2010, 20, 5050–5053.
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