Indole-based hydrazones containing a sulfonamide moiety as selective inhibitors of tumor-associated human carbonic anhydrase isoforms ix and xii

Article abstract of DOI:10.3390/ijms20092354

Novel sulfonamidoindole-based hydrazones with a 2-(hydrazinocarbonyl)-3-phenyl-1Hindole- 5-sulfonamide scaffold were synthesized and tested in enzyme inhibition assays against the tumor-associated carbonic anhydrase isoforms, hCA IX and XII, and the off-targets, hCA I and II. The compounds showed selectivity against hCA IX and XII over hCA I and II. Six compounds showed KI values lower than 10 nM against hCA IX or XII. Molecular modeling studies were performed to suggest binding interactions between the ligand and the hCA active sites.

Full text of DOI:10.3390/ijms20092354

International Journal of
Molecular Sciences
Article
Indole-Based Hydrazones Containing A Sulfonamide
Moiety as Selective Inhibitors of Tumor-Associated
Human Carbonic Anhydrase Isoforms IX and XII
1
Kübra Demir-Yazıcı 1, Silvia Bua 2 , Nurgül Mutlu Akgünes¸ , Atilla Akdemir 3
,
Claudiu T. Supuran 2 and Özlen Güzel-Akdemir 1,
*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University, 34116 Istanbul, Turkey;
kubra.demir@istanbul.edu.tr (K.D.-Y.); akgunes.nurgul@gmail.com (N.M.A.)
2
3
Department of NEUROFARBA, Sezione di Scienze Farmaceutiche Universita degli Studi di Firenze,
Sesto Fiorentino, 50019 Florence, Italy; silvia.bua@unifi.it (S.B.); claudiu.supuran@unifi.it (C.T.S.)
Computer-Aided Drug Discovery Laboratory, Department of Pharmacology, Faculty of Pharmacy,
Bezmialem Vakif University, 34093 Istanbul, Turkey; aakdemir@bezmialem.edu.tr
Correspondence: oguzel@istanbul.edu.tr
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 April 2019; Accepted: 10 May 2019; Published: 12 May 2019
Abstract: Novel sulfonamidoindole-based hydrazones with a 2-(hydrazinocarbonyl)-3-phenyl-1H-
indole-5-sulfonamide scaffold were synthesized and tested in enzyme inhibition assays against the
tumor-associated carbonic anhydrase isoforms, hCA IX and XII, and the off-targets, hCA I and II.
The compounds showed selectivity against hCA IX and XII over hCA I and II. Six compounds showed
K
I values lower than 10 nM against hCA IX or XII. Molecular modeling studies were performed to
suggest binding interactions between the ligand and the hCA active sites.
Keywords: carbonic anhydrase; sulfonamides; hydrazones; enzyme inhibition; molecular modeling
1. Introduction
Carbonic anhydrases (CA) are metalloenzymes that catalyze the reversible hydration of CO2
−
to HCO3 and H+. This simple but important reaction is widespread amongst many if not all
organisms [
contain a Zinc ion (Zn2+) at the active site that is essential for catalysis [
physiological and pathological functions, such as respiration and transporting CO2/HCO3− between
metabolizing tissues and lungs, electrolyte secretion [ ], pH regulation [ ], and biosynthetic reactions
such as gluconeogenesis, lipogenesis, ureagenesis [5] and carcinogenicity [6].
The human carbonic anhydrases (hCAs) are a member of the CAs, which are divided into
16 different isozymes (hCA I-XVI) [ ]. These enzymes share similar structure, especially in the active
site, but show different catalytic activities and tissue distributions [ ]. Cytosolic hCA isoforms I and II
(hCA I and hCA II) are widespread in the human body and are targets of clinically used diuretics [ ],
1]. Many of the six structurally different classes of CAs (i.e.,
α, β, γ, δ, ζ, and ηCAs)
2]. CAs have several important
3
4
α
7
8
9
antiglaucoma [10], drugs, and anticonvulsants [11]. In contrast, transmembrane isoforms, hCA IX and
hCA XII, which have an active site on the extracellular part of the cell membrane, are located mainly
on hypoxic tumor cells [12] and are validated drug targets for the design of anticancer agents specific
for solid hypoxic tumors [13]. Proliferation and survival of tumor cells seem to be closely related to
overexpression of both enzymes [14]. Thus, selective inhibition of hCA IX and hCA XII with well
characterized CA inhibitors (CAIs) may result in new drugs in chemotherapy.
Sulfonamides, a main class of CAIs, show their effect by binding to the Zn2+ ion of the hCA active
site. Many compounds with a sulfonamide moiety are in clinical use as CAIs or at the development
process. Most of the sulfonamides act as strong CAIs for many hCAs—including isoforms I and
Int. J. Mol. Sci. 2019, 20, 2354; doi:10.3390/ijms20092354
www.mdpi.com/journal/ijms

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II—causing a wide range of side effects. However, the design of new CAIs, which show selective
inhibition for tumor-associated isoenzymes, hCA IX and XII, and weak affinity for hCA I and II,
currently receives great attention in medicinal chemistry research [15
derivatives were investigated during the last years and have been evaluated as selective inhibitors of
several classes of carbonic anhydrases, including human/mammalian isoforms and pathogens [18 19].
–17]. Indole based sulfonamide
,
Members of our group investigated 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide for their
interaction with 12 carbonic anhydrase isoforms in the search of compounds with good inhibitory
activity against isozymes, such as CA I, II, VA, VB, VII, IX, and XII, among others [20]. Thus, we explore
here the synthesis and structure–activity relationship (SAR) for the inhibition of four CA isoforms
(hCA I, II, IX, and XII) with hydrazone derivatives of these compounds as putative CAIs.
2. Results
2.1. Chemistry
As was previously reported by our group, 3-phenyl-5-sulfonamido-1H-indole-2-carbohydrazide
was prepared from sulfanilamide as outlined in Scheme 1. After diazotization of the sulfanilamide,
condensation of diazonium salt with ethyl 2-benzylacetoacetate led to an intermediate, which was
cyclized in acidic medium with formation of the ethyl ester derivative of , which was converted to
by treatment with hydrazine [20]. Further treatment of with an appropriate carbonyl compound
(ketone or aldehyde) yielded hydrazone derivatives 4-24 (Scheme 1).
1
2
3
3
3
Scheme 1. Reagents and conditions: (i
) NaNO2, HCl, 0 ◦C; (ii) KOH, 0 ◦C; (iii) 37% HCl, reflux, 4 h;
(iv) H2NNH2.H2O, EtOH, reflux, 6 h; (v) reflux, 6–12 h.
Newly synthesized hydrazones were characterized with melting points and spectral analyses.
The IR spectra of compounds 4-24 showed NH stretching bands of the hydrazide-hydrazone group
and sulfonamide moiety at the indole ring at 3423–3138 cm−1. The carbonyl functionalities of the
new compounds were confirmed by strong C=O stretching bands observed in the 1643–1685 cm−1
,
while compound
3
showed a band of 1627 cm−1, as expected. Asymmetric and symmetric SO2
stretching vibrations of the sulfonamide group had absorption bands in the 1344–1311 cm−1 and
1176–1147 cm−1 [21,22].

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l
The H-NMR spectrum of compounds displayed the N-H, the C4-H, the C6-H, and the C7-H
protons of the indole ring at 12.39–12.14 ppm (for 9, the second indole ring N-H proton was at 11.59 ppm)
as a singlet and 8.85–7.15 ppm, 7.75–7.71, and 7.65–7.61 ppm, respectively. The N-H protons of the
hydrazide structure exhibited the expected singlets at the 11.75–9.01 ppm [19]. The SO2NH2 protons
had signals at 7.21–7.15 ppm. For compounds 4-11, the azomethine protons resonated at 9.24–8.09 ppm
except 10. A broad resonance with 2H integration value at 8.21 and 8.24 ppm was assigned to the two
azomethine protons of 10. All the other protons were observed in the expected regions. In the 13
C
NMR spectrum of compounds, carbon atoms of the hydrazide carbonyl (CONH) and the hydrazone
(C=N) groups were observed at in the order of 167.18–157.75 and 157.50–143.19 ppm.
2.2. Enzyme Inhibition Assays
All synthesized compounds were tested in enzyme inhibition assays against the widespread and
cytosolic hCA I and II enzymes and the tumor-associated and transmembrane hCA IX and XII enzymes
with extracellular catalytic domains (Table 1, Figure 1). The compounds showed selectivity for hCA IX
and XII over hCA I and II (Table 1, Figure 1). For hCA IX, 14 of the tested compounds had KI values
smaller than 100 nM, including one compound with a KI value of less than 10 nM (i.e., compound 23).
Of the 14 compounds, seven had a similar or lower KI value compared to acetazolamide (AAZ).
For hCA XII, five of the tested compounds had a similar or lower KI value compared to acetazolamide.
Compound 23 was selective for hCA IX with ~seven-fold selectivity over hCA XII and at least ~33-fold
selectivity over the off-targets, hCA I and II. Additionally, this compound had a lower KI value
compared to acetazolamide (AAZ).
Compound 17 had a cyclohexyl moiety, which was not very well tolerated (Table 1, Figure 1).
Adding flexible aliphatic substituents to the cyclohexyl moiety increased the KI value of the ligand
for hCA IX, while adding an aromatic phenyl or bulky tert-butyl substituent (compounds 21 and 22,
respectively) decreased the KI values (Table 1, Figure 1). A cyclopentyl instead of a cyclohexyl moiety
was also not tolerated.
Compound 18, the analog of compound 23 without a cationic nitrogen atom, showed a ~45-fold
higher KI value compared to compound 23. This may indicate that a cationic charge was favorable in
the ligand binding interactions.
The compounds had, in general, lower KI values for hCA XII, with 18 compounds showing KI
smaller than 100 nM, including five compounds with KI smaller than 10 nM. Compound
lowest measured KI value for hCA XII (KI: 1.4 nM).
7 had the
The 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide was previously tested by members
of our group against the tumor-associated hCA IX and XII and the off-targets, hCA I and II [20].
Compared to this previous compound, the newly synthesized hydrazon compounds had higher
selectivity for hCA IX/XII compared to the off-targets.
Table 1. Inhibition data of hCA I, II, IX, XII with compounds 4-24 and the standard inhibitor
acetazolamide (AAZ) by a stopped flow CO2 hydrase assay.
*
KI (nM)
Cpd.
hCA I
hCA II
hCAIX
hCA XII
R
R1
4
5
6
7
8
6667.5
9408.7
9572
8592.1
3134.9
>10,000
7479.2
4056.8
9558.6
>10,000
>10,000
>10,000
8906.7
3487.4
>10,000
9369.7
47.4
48.4
44.3
35.8
15.8
206.8
32.9
12.9
47.9
4.5
75.1
64.4
1.4
H
H
H
H
H
H
H
H
CH3
Thiophene-2-yl
5-Br-thiophene-2-yl
1-CH3-pyrrol-2-yl
pyridine-3-yl
pyridine-4-yl
indole-3-yl
H
6.6
9
77.3
23.8
62.6
87.9
10
11
12
5866.8
8498.4
CH3
CH3

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Table 1. Cont.
*
KI (nM)
Cpd.
hCA I
hCA II
hCAIX
hCA XII
R
R1
13
14
15
16
17
18
19
20
21
22
23
24
AAZ
9619.6
9246.3
8652.3
8029.7
6621.8
6562.3
5960.5
5867.9
8942.5
1282.7
795.8
830.7
3380.8
309
42.1
43.7
187.8
476.1
191
418.9
475.3
347.7
32.3
17.4
9.2
92.4
9.4
CH3
C2H5
CH3
C2H5
C2H5
isobuthyl
27.9
97.2
86.2
139.1
219.7
153.6
14.8
26.6
62.9
9.4
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
7384.7
cyclopentyl
cyclohexyl
4-methylcyclohexyl
4-ethylcyclohexyl
4-propylcyclohexyl
4-phenylcyclohexyl
4-tert-buthylcyclohexyl
1-methylpiperidin-4-yl
1-benzylpiperidine-4-yl
372.4
12.5
11.7
25
250
5.7
-
-
* Mean from three different assays by a stopped flow technique (errors were in the range of 5–10 % of the reported
values). hCA= human carbonic anhydrases, AAZ= acetazolamide.
A
B
Figure 1.
(A) New 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide based hydrazone
derivatives (substitutions are shown in Table 1). (B) Bar-graph showing the log KI values of AAZ
(acetazolamide) and the tested compounds.
2.3. Molecular Modeling Studies of hCA IX
Compound 23 displayed the lowest KI value for hCA IX of 9.2 nM. The sulfonamide moiety
formed direct interactions with the Zn2+ ion (tetrahedral orientation) and the backbone of Thr199
(Figure 2; 23 pose in orange). The indole group of the ligand was situated between Gln92 and Leu198.
The side chain hydroxyl group of Thr200 pointed with its hydrogen atom towards the centroid of
the ligand’s phenyl group to interact with the π-cloud. A hydrogen bond was formed between the
ligand’s carbonyl group and the side chain of Gln67. The cationic aliphatic ring of the ligand was
located between Gln67, Leu91, and Val131. The latter cationic group did not form any interactions
with the active site residues. Detailed investigation of the ligand’s docked pose and the active site
suggested that the ligand cationic aliphatic group could adopt an extended conformation towards
Asp132 (Figure 1; 23 pose in purple and Asp132 rotamer in green). The latter residue could adopt
another rotameric conformation that reached for the ligand’s cationic group, and both a hydrogen
bond and an electrostatic interaction could have occurred (distance cationic nitrogen of the ligand and
the anionic oxygen atom of Asp132 2.750 Å). Such an interaction would be consistent with the enzyme
inhibition data for hCA IX in which 23 and 24, compounds with a cationic group, had the lowest KI
values. Compound 18 was an analog of 24 without the cationic nitrogen atom with a much higher KI
value. Additional analogs such as 16, 17, and 19–21 similarly had higher KI values compared to 23.

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Figure 2. The docked pose of compound 23 (orange and purple) in the active site of hCA IX (3iai).
Hydrogen bonds and interactions with the zinc ion are indicated in red dashed lines. H-arene interactions
are indicated in yellow dashed lines. The zinc ion is indicated as a turquoise sphere. The Asp132 rotamer
that was able to form a hydrogen bond with the ligand is indicated in green sticks.
2.4. Molecular Modeling Studies of hCA XII
The lowest KI value for hCA XII was measured for compound
7 (KI: 1.4 nM). Again, the
sulfonamide moiety formed direct interactions with the Zn2+ ion (tetrahedral orientation) and the
backbone of Thr199 (Figure 3). The phenyl and the pyridine groups of the ligands formed H-arene
interactions with the hydrogen atoms of Asn62. Further hydrophobic interactions were formed with
the side chains of His94, Val121, and Leu198. No hydrogen bonds were formed between the ligand
and the active site residues. However, the side chain of Gln92 could adopt a conformation in which a
hydrogen bond with the ligand carbonyl group was possible (Figure 3).
In general, the docked poses of the compounds, especially the placement of the sulfonamide and
the indole parts of the ligands, were similar due to structural similarity between the compounds and
the requirements to form an interaction between the sulfonamide and the zinc ion.
Compounds 18
interactions with the active site. This may explain the higher KI values for these ligands. Remarkably,
compound 21 had a lower I value even though it also had a bulky hydrophobic substituent. The phenyl
, 19, and 20 had bulky hydrophobic groups that could not form hydrophobic
K
group of the ligand may have formed hydrophobic interactions with the side chain of Asn69 and a
Thr91 rotamer (with the methyl group pointing to the ligand instead of the hydroxyl group; Figure 4).
It seems this interaction was not possible for compounds 18, 19, and 20.
Figure 3. The docked pose of compound 7 (turquoise) in the active site of hCA XII (1jd0). Hydrogen
bonds and interactions with the zinc ion are indicated in red dashed lines. H-arene interactions are
indicated in yellow dashed lines. The zinc ion is indicated as a turquoise sphere. The Gln92 rotamer
that was able to form a hydrogen bond with the ligand is indicated in green sticks.

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Figure 4. The docked pose of compound 20 (orange) and 21 (purple) in the active site of hCA XII
(1jd0). Hydrogen bonds and interactions with the zinc ion are indicated in red dashed lines. H-arene
interactions are indicated in yellow dashed lines. The zinc ion is indicated as a turquoise sphere.
The Thr91 rotamer that was able to form an H-arene interaction with 21 is indicated in green sticks.
2.5. Prediction of ADMET Properties
The potential mutagenicity [23], the reactivity [24] and Lipinski’s parameters (weight, hydrogen
bond donor, and acceptor count) of the compounds were calculated using the MOE software package
(v2018.0101, Chemical Computing Group Inc., Montreal), and the LogP value [25] was calculated using
the ALogPs 2.1 webserver [26]. No violation of Lipinski’s rule of five was found [27
the compounds may have passed cell membranes by passive diffusion. Compounds
were not predicted to be mutagenic, but of these four compounds, only compound was not predicted
,
28], indicating that
7,
20, 21, and 23
7
to be reactive. It should be noted, however, that these were theoretical predictions, and experimental
validation is still needed.
3. Materials and Methods
Melting points were estimated with a Büchi B-540 (BÜCHI Labortechnik AG, Flawil, Switzerland)
melting point apparatus in open capillaries and are presented as uncorrected. Elemental analyses
were performed on a Thermo Finnigan Flash EA 1112 (Thermo Electron GmbH, Bremen, Germany)
elemental analyzer. IR spectra were recorded on KBr discs using a Perkin-Elmer (Waltham, MA, USA)
1
Model 1600 FT-IR spectrometer. H NMR, 13C-NMR (decoupled), and HSQC-2D spectra were obtained
on Varian UNITY INOVA 500, Varian Mercury (Agilent, Palo Alto, CA, USA) 400 MHz FT-NMR
spectrophotometers using DMSO-d6. The mass spectra were taken on a Waters ZQ micromass LC-MS
spectrometer (Waters Corporation, Milford, MA, USA) by using ESI (+) method.
3.1. Synthetic Procedures
3.1.1. 2-Benzyl-2-[N-(4-sulfonamidophenyl)hydrazono]ethanoate (1).
To a solution of 0.01 mole sulfanilamide in 4 mL of 37% HCl, 10 mL of 8% NaNO2 aqueous
solution was added dropwise at 0 ◦C. The solution containing the diazonium salt was poured into an
ice-cold mixture of 2.3 g (a little excess of 0.01 mole) ethyl 2-benzylacetoacetate, 10 mL of C2H5OH,
20 mL of H2O, and 2.7 g of KOH. The mixture was kept cold overnight. The hydrazone, produced as
an oil, was separated, dissolved in (C2H5)2O, washed with H2O, and dried over anhydrous Na2SO4.
(C2H5)2O was distilled, and the oily residue was treated with 5 mL of 37% HCl and set aside for 5 h at
room temperature. The resulting solid substance was recrystallized from EtOH.

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3.1.2. Ethyl 5-(aminosulfonyl)-3-phenyl-1H-indole-2-carboxylate (2).
A mixture of 0.01 mole ethyl 2-benzyl-2-[N-(4-sulfonamidophenyl)hydrazono]ethanoate and
about 10 mL of 37% HCl was heated on a water bath for 4 h, cooled, and poured into 100 mL of H2O.
The crude product was filtered, washed with H2O, and recrystallized from EtOH.
3.1.3. 2-(hydrazinylcarbonyl)-3-phenyl-1H-indole-5-sulfonamide (3).
For this compound, 3.45 g (0.01 mole) ethyl 5-(aminosulfonyl)-3-phenyl-1H-indole-2-carboxylate
was dissolved in 20 mL of C2H5OH, then 4 mL of H2NNH2-H2O was added and refluxed for 6 h,
cooled, and kept cold overnight. The resulting crystals were filtered off, washed with (C2H5)2O, and
recrystallized from EtOH /DMF.
3.1.4. General procedure for the synthesis of 3-phenyl-5-sulfamoyl-N’-[(heteroaryl)methylidene]-
1H-indole-2-carbohydrazide/3-phenyl-5-sulfamoyl-N’-[(non)substituedcycloalkylidene/alkylidene]-
1H-indole-2-carbohydrazide derivatives (4-24).
A mixture of 3-phenyl-5-sulfonamidoindole-2-carbohydrazide (3) (0.005 mole), an appropriate
aldehyde/heteroaromatic aldehyde/ketone/cyclic ketone (0.005 mole), was refluxed in 20 mL absolute
ethanol for 6–12 h. The precipitate obtained was purified either by recrystallization from ethanol or by
washing with hot ethanol.
3.1.5. 3-Phenyl-2-{[2-(thiophene-2-ylmethylidene)hydrazinyl]carbonyl}-1H-indole-5-sulfonamide (4)
Yellow powder (25%); mp 309–310 ◦C; IR(KBr):
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 7.13 (1H, s, thiophene C4-H), 7.20 (2H, s, SO2NH2), 7.39
υ 3400, 3329, 3250 (NH), 1654 (C=O); 1319, 1147
δ
(1H, br s, thiophene C3-H), 7.44 (1H, br s, thiophene C5-H), 7.50-7.57 (5H, m, 3-phenyl C-H), 7.64 (1H,
d, J=8.79 Hz, indole C7-H), 7.74 (1H, d, J=8.79 Hz, indole C6-H), 8.14 (1H, s, indole C4-H), 8.31 (1H, s,
N=CH), 11.44 (1H, s, CONH), 12.35 (1H, s, indole NH). 13C-NMR (HSQC-2D, DMSO-d6, 125 MHz)
δ
(ppm): 113.19 [ind. C7 - 7.64 (1H, d, J=8.79 Hz, indole C7-H)], 119.06 [ind. C4 - 8.14 (1H, s, indole
C4-H)], 121.79 [ind. C6 - 7.74 (1H, d, J=8.79 Hz, indole C6-H)], 127.59 [thiophene C3 - 7.39 (1H, br s,
thiophene C3-H),], 128.32 [thiophene C4 - 7.13 (1H, s, thiophene C4-H)], 129.05, 129.77, 130,16 [3-Phenyl
C - 7.50-7.57 (5H, m, 3-phenyl C-H)], 131.88 [thiophene C5 - 7.44 (1H, br s, thiophene C5-H)], 143.19
[N=CH - 8.31 (1H, s, N=CH)]. Anal. Calcd. for C20H16N4O3S2 (424.496): C, 56.59; H, 3.80; N, 13.20; S,
15.11. Found: C, 56.27; H, 3.60; N, 11.61; S, 15.43.
3.1.6. 2-({2-[(5-Bromothiophene-2-yl)methylidene]hydrazinyl}carbonyl)-3-phenyl-1H-indole-5-sulfonamide (5)
Yellow powder (30%); mp 272–273 ◦C; IR(KBr):
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 7.20 (2H, s, SO2NH2), 7.26 (1H, br s, thiophene C4-H),
υ 3423, 3300, 3217 (NH), 1654 (C=O); 1334, 1153
δ
7.31 (1H, br s, thiophene C3-H), 7.40 (1H, br s, 3-phenyl C-H), 7.52 (4H, br s, 3-phenyl C-H), 7.64 (1H, d,
J=8.78 Hz, indole C7-H), 7.73 (1H, dd, J=9.04, 1.46 Hz, indole C6-H), 8.13 (1H, s, indole C4-H), 8.24 (1H,
s, N=CH), 11.52 (1H, s, CONH), 12.32 (1H, s, indole NH). Anal. Calcd. for C20H15BrN4O3S2 (503.392):
C, 47.72; H, 3.00; N, 11.13; S, 12.74. Found: C, 47.82; H, 3.30; N, 11.41; S, 12.90.
3.1.7. 2-({2-[(1-Methyl-1H-pyrrol-2-yl)methylidene]hydrazinyl}carbonyl)-3-phenyl-1H-indole-5-sulfonamide (6)
Yellow powder (40%); mp 298–299 ◦C; IR(KBr): υ 3419, 3313, 3253, 3213 (NH), 1645 (C=O); 1307,
1
1157 (S=O); H NMR (DMSO-d6/500 MHz
δ (ppm): 3.84 (3H, s, pyrrol 1-CH3), 6.10 (1H, s, pyrrol
C3-H), 6.48 (1H, s, pyrrol C4-H), 6.98 (1H, s, pyrrol C5-H), 7.20 (2H, s, SO2NH2), 7.41 (2H, d, J=6.83
Hz, 3-phenyl C-H), 7.51-7.56 (3H, m, 3-phenyl C-H), 7.64 (1H, d, J=8.78 Hz, indole C7-H), 7.74 (1H, d,
J=8.30 Hz, indole C6-H), 8.10 (1H, s, indole C4-H), 8.13 (1H, s, N=CH), 11.13 (1H, s, CONH), 12.31 (1H,
s, indole NH). Anal. Calcd. for C21H19N5O3S (421.472): C, 59.84; H, 4.54; N, 16.62; S, 7.61. Found: C,
59.67; H, 4.62; N, 16.81; S, 7.83.

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3.1.8. 3-Phenyl-2-{[2-(pyridine-3-ylmethylidene)hydrazinyl]carbonyl}-1H-indole-5-sulfonamide (7)
Ivory powder (60%); mp 287.5–287.8 ◦C; IR(KBr):
3360, 3275, 3190 (NH), 1643 (C=O); 1340, 1159
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 7.19 (2H, s, SO2NH2), 7.32-7.53 (6H, m, 3-phenyl C-H
υ
δ
and pyridine C5-H), 7.65 (1H, d, J=8.78 Hz, indole C7-H), 7.74 (1H, d, J=8.30 Hz, indole C6-H), 8.14
(2H, br s, indole C4-H and N=CH), 8.48 (1H, br s, pyridine C4-H), 8.60 (1H, s, pyridine C6-H), 8.82
(1H, s, pyridine C2-H), 11.64 (1H, s, CONH), 12.38 (1H, s, indole NH). Anal. Calcd. for C21H17N5O3S
(419.456): C, 60.13; H, 4.09; N, 16.70; S, 7.64. Found: C, 59.80; H, 3.93; N, 16.61; S, 7.43.
3.1.9. 3-Phenyl-2-{[2-(pyridine-4-ylmethylidene)hydrazinyl]carbonyl}-1H-indole-5-sulfonamide (8)
Yellow powder (40%); mp 271.5–271.7 ◦C; IR(KBr):
1151 (S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 7.21 (2H, s, SO2NH2), 7.29-7.58 (7H, m, 3-phenyl
υ 3236, 3178, 3163 (NH), 1666 (C=O); 1321,
δ
C-H and pyridine C3,5-H), 7.65 (1H, d, J=8.30 Hz, indole C7-H), 7.75 (1H, dd, J=8.78, 1.47 Hz, indole
C6-H), 8.09 (1H, s, indole C4-H), 8.15 (1H, br s, N=CH), 8.62 (2H, br s, pyridine C2,6-H), 11.75 (1H, s,
CONH), 12.38 (1H, s, indole NH). Anal. Calcd. for C21H17N5O3S (419.456): C, 60.13; H, 4.09; N, 16.70;
S, 7.64. Found: C, 60.33; H, 4.35; N, 16.91; S, 7.93.
3.1.10. 3-Phenyl-2-{[2-(1H-indole-3-ylmethylidene)hydrazinyl]carbonyl}-1H-indole-5-sulfonamide (9)
Yellow crystal (20%); mp 264–265 ◦C; IR(KBr):
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 7.15-7.17 (4H, m, 2.indole C5,6-H and SO2NH2), 7.30-7.45
υ 3373, 3313, 3211 (NH), 1643 (C=O); 1319, 1151
δ
(2H, m, 3-phenyl C4-H and 2.indole C7-H), 7.51 (2H, t, J=7.81 Hz, 3-phenyl C3,5-H), 7.58 (2H, d, J=7.32
Hz, 3-phenyl C2,6-H), 7.64 (1H, d, J=8.78 Hz, indole C7-H), 7.70 (1H, dd, J=8.78, 1.46 Hz, indole C6-H),
7.79 (1H, d, J=2.93 Hz, 2.indole C2-H), 8.13 (1H, s, indole C4-H), 8.25 (1H, s, N=CH), 8.26 (1H, d, J=7.81
Hz, 2. indole C4-H), 11.07 (1H, s, CONH), 11.59 (1H, s, 2. indole NH), 12.34 (1H, s, indole NH). Anal.
Calcd. for C24H19N5O3S (457.504): C, 63.01; H, 4.19; N, 15.31; S, 7.01. Found: C, 59.87; H, 4.50; N, 15.61;
S, 7.33.
3.1.11. 2-[(2-Methylidenehydrazinyl)carbonyl]-3-phenyl-1H-indole-5-sulfonamide (10)
White powder (30%); mp 248.5–248.8 ◦C; IR(KBr):
υ
3244 (NH), 1645 (C=O); 1323, 1149 (S=O); 1H
NMR (DMSO-d6/500 MHz (ppm): 7.17 (2H, s, SO2NH2), 7.35-7.49 (5H, m, 3-phenyl C-H), 7.62 (1H, d,
δ
J=8.21 Hz, indole C7-H), 7.70 (1H, d, J=8.21 Hz, indole C6-H), 8.10 (1H, s, indole C4-H), 8.21, 8.24 (2H,
2d, J=3.52 Hz, N=CH2), 11.26 (1H, s, CONH), 12.14 (1H, s, indole NH). Anal. Calcd. for C16H14N4O3S
(342.372): C, 56.13; H, 4.12; N, 16.36; S, 9.37. Found: C, 56.50; H, 3.93; N, 16.60; S, 9.67.
3.1.12. 2-[(2-Ethylidenehydrazinyl)carbonyl]-3-phenyl-1H-indole-5-sulfonamide (11)
Ivory powder (27%); mp 250–250.1 ◦C; IR(KBr):
1H NMR (DMSO-d6/500 MHz
(ppm): 1.88 (3H, d, J=3.90 Hz, -CH3), 7.18 (2H, s, SO2NH2), 7.34-7.44
υ 3302, 3221 (NH), 1670 (C=O); 1323, 1145 (S=O);
δ
(2H, m, 3-phenyl C4-H and N=CH), 7.46-7.54 (4H, m, 3-phenyl C2,3,5,6-H), 7.61 (1H, d, J=8.30 Hz, indole
C7-H), 7.75 (1H, d, J=8.78 Hz, indole C6-H), 8.10 (1H, d, J=1.95 Hz, indole C4-H), 11.00 (1H, s, CONH),
12.31 (1H, s, indole NH). 13CNMR (proton decoupled, DMSO-d6, 150 MHz)
δ (ppm): 56.15 (-CH3),
112.69 (ind. C7), 118.64 (ind. C4), 119.20 (ind. C3), 121.26 (ind. C6), 125.46 (ind. C3a), 127.17 (3-Phenyl
C4), 128.65 (3-Phenyl C3/5), 129.19 (ind. C2), 129.64 (3-Phenyl C2/6), 133.03 (ind. C5), 136.74 (3-Phenyl
C1), 139.05 (ind. C7a), 148.67 (N=C), 157.75 (CONH). Anal. Calcd. for C17H16N4O3S (356.398): C, 57.29;
H, 4.52; N, 15.72; S, 9.00. Found: C, 56.96; H, 4.13; N, 16.05; S, 9.37. m/z ESI (positive) 357.36 (M+H)+.
3.1.13. 3-Phenyl-2-{[2-(propan-2-ylidene)hydrazinyl]carbonyl}-1H-indole-5-sulfonamide (12)
◦
White powder (30%); mp 287.9–288 C; IR(KBr):
υ
3334, 3305, 3217 (NH), 1668 (C=O); 1325,
1147 (S=O); 1H NMR (DMSO-d6/500 MHz
δ
(ppm): 1.27 (3H, s, -CH3), 1.89 (3H, s, -CH3), 7.17 (2H, s,
SO2NH2), 7.45-7.56 (5H, m, 3-phenyl C-H), 7.62 (1H, d, J=8.30 Hz, indole C7-H), 7.72 (1H, dd, J=8.30,
1.95 Hz, indole C6-H), 7.91 (1H, s, indole C4-H), 9.24 (1H, s, CONH), 12.39 (1H, s, indole NH). Anal.

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Calcd. for C18H18N4O3S (370.425): C, 58.36; H, 4.90; N, 15.12; S, 8.66. Found: C, 58.76; H, 5.13; N, 15.05;
S, 9.01.
3.1.14. 2-{[2-(Butan-2-ylidene)hydrazinyl]carbonyl}-3-phenyl-1H-indole-5-sulfonamide (13)
White powder (78%); mp 285.5–285.6 ◦C; IR(KBr):
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 0.93-1.04 (3H, m, -CH3), 1.26 (3H, s, -CH3), 2.20 (2H,
υ 3360, 3304, 3224 (NH), 1680 (C=O); 1315, 1151
δ
d, J=4.88 Hz, -CH2-), 7.17 (2H, s, SO2NH2), 7.45-7.57 (5H, m, 3-phenyl C-H), 7.63 (1H, d, J=8.79 Hz,
indole C7-H), 7.71 (1H, dd, J=8.79, 1.46 Hz, indole C6-H), 7.91 (1H, s, indole C4-H), 9.19 (1H, s, CONH),
12.35 (1H, s, indole NH). Anal. Calcd. for C19H20N4O3S (384.452): C, 59.36; H, 5.24; N, 14.57; S, 8.34.
Found: C, 59.56; H, 5.36; N, 14.15; S, 8.71.
3.1.15. 2-{[2-(Pentan-3-ylidene)hydrazinyl]carbonyl}-3-phenyl-1H-indole-5-sulfonamide (14)
White powder (60%); mp 288.2–288.3 ◦C; IR(KBr):
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 0.63 (3H, s, -CH3), 1.00 (3H, s, -CH3), 1.60 (2H, d, J=6.04
υ 3356, 3300, 3221 (NH), 1680 (C=O); 1319, 1151
δ
Hz, -CH2-), 2.20 (2H, d, J=6.04 Hz, -CH2-), 7.15 (2H, s, SO2NH2), 7.46-7.58 (5H, m, 3-phenyl C-H), 7.62
(1H, d, J=8.78 Hz, indole C7-H), 7.71 (1H, dd, J=8.23, 1.65 Hz, indole C6-H), 7.85 (1H, s, indole C4-H),
9.20 (1H, s, CONH), 12.39 (1H, s, indole NH). Anal. Calcd. for C20H22N4O3S (398.478): C, 60.28; H,
5.56; N, 14.06; S, 8.05. Found: C, 59.96; H, 5.16; N, 14.40; S, 8.36.
3.1.16. 2-{[2-(4-Methylpentan-2-ylidene)hydrazinyl]carbonyl}-3-phenyl-1H-indole-5-sulfonamide (15)
White powder (50%); mp 255.1–255.2 ◦C; IR(KBr):
1151 (S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 0.83 (6H, d, J=5.49 Hz, -CH(CH3)2), 1.24 (3H, s,
υ 3352, 3321, 3265 (NH), 1674 (C=O); 1332,
δ
-CH3), 1.84-1.88 (1H, m, -CH-), 2.05 (2H, d, J=5.49 Hz, -CH2-), 7.16 (2H, s, SO2NH2), 7.47-7.56 (5H, m,
3-phenyl C-H), 7.62 (1H, d, J=8.78 Hz, indole C7-H), 7.72 (1H, dd, J=8.78, 1.65 Hz, indole C6-H), 7.91
(1H, s, indole C4-H), 9.20 (1H, s, CONH), 12.36 (1H, s, indole NH). Anal. Calcd. for C21H24N4O3S
(412.505): C, 61.14; H, 5.86; N, 13.58; S, 7.77. Found: C, 60.83; H, 5.50; N, 13.35; S, 7.56.
3.1.17. 2-[(2-Cyclopentylidenehydrazinyl)carbonyl]-3-phenyl-1H-indole-5-sulfonamide (16)
White powder (84%); mp 313.1–313.2 ◦C; IR(KBr):
1153 (S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 1.50-1.65 (6H, m, -cyclopentyl C-H), 2.30 (2H,
υ 3336, 3234, 3138 (NH), 1658 (C=O); 1344,
δ
m, -cyclopentyl C-H), 7.17 (2H, s, SO2NH2), 7.50-7.59 (5H, m, 3-phenyl C-H), 7.61 (1H, d, J=8.78 Hz,
indole C7-H), 7.71 (1H, dd, J=8.78, 0.97 Hz, indole C6-H), 7.89 (1H, s, indole C4-H), 9.01 (1H, s, CONH),
12.39 (1H, s, indole NH). 13CNMR (proton decoupled, DMSO-d6, 150 MHz)
δ (ppm): 24.08, 24.11
(cyclopentyl C3/4), 26.62, 32.59 (cyclopentyl C2/5), 112.64 (ind. C7), 118.53 (ind. C4), 121.35 (ind. C6),
126.16 (ind. C3a), 127.93 (3-Phenyl C4), 128.90 (3-Phenyl C3/5), 129.19 (ind. C2), 130.19 (3-Phenyl C2/6),
132.85 (ind. C5), 136.35 (3-Phenyl C1), 136.58 (ind. C7a), 156.90 (N=C), 167.18 (CONH). Anal. Calcd.
for C20H20N4O3S (396.462): C, 60.59; H, 5.08; N, 14.13; S, 8.09. Found: C, 60.73; H, 5.10; N, 13.85; S,
7.86. m/z ESI (positive) 396.40 (M+H)+.
3.1.18. 2-[(2-Cyclohexylidenehydrazinyl)carbonyl]-3-phenyl-1H-indole-5-sulfonamide (17)
White powder (80%); mp 285.5–285.6 ◦C; IR(KBr):
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 1.34 (2H, s, -cyclohexyl C-H), 1.49 (2H, s, - cyclohexyl
υ 3358, 3296, 3147 (NH), 1676 (C=O); 1317, 1149
δ
C-H), 1.60 (4H, s, -cyclohexyl C-H), 2.28 (2H, s, -cyclohexyl C-H), 7.16 (2H, s, SO2NH2), 7.45-7.57 (5H, m,
3-phenyl C-H), 7.61 (1H, d, J=8.79 Hz, indole C7-H), 7.72 (1H, dd, J=7.32, 1.46 Hz, indole C6-H), 7.94 (1H,
s, indole C4-H), 9.52 (1H, s, CONH), 12.36 (1H, s, indole NH). 13CNMR (proton decoupled, DMSO-d6,
150 MHz) δ (ppm): 24.80 (cyclohexyl C4), 25.50, 26.26 (cyclohexyl C3/5), 26.56, 34.69 (cyclohexyl C2/6),
112.56 (ind. C7), 118.46 (ind. C4), 121.18 (ind. C6), 125.95 (ind. C3a), 127.59(3-Phenyl C4), 128.93
(3-Phenyl C3/5), 129.40 (ind. C2), 130.10 (3-Phenyl C2/6), 132.82 (ind. C5), 136.29 (3-Phenyl C1), 136.64

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(ind. C7a), 157.26 (N=C), 161.59 (CONH). Anal. Calcd. for C21H22N4O3S (410.489): C, 61.44; H, 5.40; N,
13.65; S, 7.81. Found: C, 61.83; H, 5.17; N, 13.86; S, 7.46. m/z ESI (positive) 411.42 (M+H)+.
3.1.19. 2-{[2-(4-Methylcyclohexylidene)hydrazinyl]carbonyl}-3-phenyl-1H-indole-5-sulfonamide (18)
White powder (75%); mp 295.6–295.7 ◦C; IR(KBr):
1147 (S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 0.87 (3H, d, J=5.37 Hz, 4-CH3), 1.04-1.14 (1H, s,
υ 3350, 3309, 3165 (NH), 1666 (C=O); 1315,
δ
-cyclohexyl C-H), 1.47-1.58 (4H, m, - cyclohexyl C-H), 1.72-1.80 (2H, m, -cyclohexyl C-H), 2.11-2.22 (1H,
m, -cyclohexyl C-H), 2.31 (1H, d, J=12.20 Hz, -cyclohexyl C-H), 7.16 (2H, s, SO2NH2), 7.42-7.56 (5H, m,
3-phenyl C-H), 7.61 (1H, d, J=8.79 Hz, indole C7-H), 7.71 (1H, dd, J=8.79 Hz, indole C6-H), 7.93 (1H, s,
indole C4-H), 9.51 (1H, s, CONH), 12.35 (1H, s, indole NH). Anal. Calcd. for C22H24N4O3S (424.515):
C, 62.24; H, 5.70; N, 13.20; S, 7.55. Found: C, 61.98; H, 5.63; N, 12.85; S, 7.16.
3.1.20. 2-{[2-(4-Ethylcyclohexylidene)hydrazinyl]carbonyl}-3-phenyl-1H-indole-5-sulfonamide (19)
White powder (74%); mp 282.4–282.5 ◦C; IR(KBr):
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 0.85 (3H, t, J=7.32 Hz, 4-CH2CH3), 1.16 (1H, t, J=7.32
υ 3348, 3311, 3176 (NH), 1666 (C=O); 1315, 1147
δ
Hz, -cyclohexyl C-H), 1.21 (2H, t, J=6.83 Hz, 4-CH2CH3), 1.35 (1H, br s, -cyclohexyl C-H), 1.49 (1H,
t, J=13.67 Hz, -cyclohexyl C-H), 1.62 (2H, d, J=12.20 Hz, -cyclohexyl C-H), 1.75 (1H, d, J=13.67 Hz,
-cyclohexyl C-H), 1.85 (1H, d, J=9.27 Hz, -cyclohexyl C-H), 2.14 (1H, t, J=11.71 Hz, -cyclohexyl C-H),
2.32 (1H, d, J=12.20 Hz, -cyclohexyl C-H), 7.17 (2H, s, SO2NH2), 7.37-7.57 (5H, m, 3-phenyl C-H), 7.62
(1H, d, J=8.79 Hz, indole C7-H), 7.72 (1H, dd, J=8.79 Hz, indole C6-H), 7.93 (1H, s, indole C4-H), 9.50
(1H, s, CONH), 12.37 (1H, s, indole NH). Anal. Calcd. for C23H24N4O3S (424.515): C, 62.24; H, 5.70; N,
13.20; S, 7.55. Found: C, 62.55; H, 5.38; N, 13.63; S, 7.47.
3.1.21. 2-{[2-(4-Propylcyclohexylidene)hydrazinyl]carbonyl}-3-phenyl-1H-indole-5-sulfonamide (20)
White powder (46%); mp 249.7–250 ◦C; IR(KBr):
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 0.86 (3H, t, J=7.32 Hz, 4-CH2CH2CH3), 1.06 (1H, t, J=7.32
υ 3354, 3331, 3236 (NH), 1662 (C=O); 1342, 1143
δ
Hz, -cyclohexyl C-H), 1.15 (2H, d, J=6.34 Hz, 4-CH2CH2CH3), 1.28 (2H, q, J=7.32 Hz, 4-CH2CH2CH3),
1.45-1.51 (3H, m, - cyclohexyl C-H), 1.60 (1H, d, J=12.20 Hz, -cyclohexyl C-H), 1.74 (1H, d, J=13.67 Hz,
-cyclohexyl C-H), 1.84 (1H, d, J=14.00 Hz, -cyclohexyl C-H), 2.14 (1H, t, J=10.74 Hz, -cyclohexyl C-H),
2.30-2.36 (1H, m, -cyclohexyl C-H), 7.16 (2H, s, SO2NH2), 7.36-7.57 (5H, m, 3-phenyl C-H), 7.61 (1H, d,
J=8.79 Hz, indole C7-H), 7.72 (1H, d, J=8.79 Hz, indole C6-H), 7.93 (1H, s, indole C4-H), 9.50 (1H, s,
CONH), 12.36 (1H, s, indole NH). Anal. Calcd. for C24H28N4O3S (452.569): C, 63.69; H, 6.24; N, 12.38;
S, 7.09. Found: C, 63.45; H, 6.47; N, 12.78; S, 7.49.
3.1.22. 2-{[2-(4-Phenylcyclohexylidene)hydrazinyl]carbonyl}-3-phenyl-1H-indole-5-sulfonamide (21)
White powder (80%); mp 312.8–313 ◦C; IR(KBr):
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 1.28-1.41 (1H, m, -cyclohexyl C-H), 1.58-1.77 (3H, m,
υ 3358, 3319, 3280 (NH), 1670 (C=O); 1338, 1159
δ
-cyclohexyl C-H), 1.87 (1H, d, J=13.18 Hz, -cyclohexyl C-H), 1.96 (1H, br s, -cyclohexyl C-H), 2.26-2.39
(1H, m, -cyclohexyl C-H), 2.46 (1H, d, J=13.18 Hz, -cyclohexyl C-H), 2.78 (1H, m, -cyclohexyl C-H),
7.17 (2H, s, SO2NH2), 7.19-7.21 (3H, m, cyclohexyl 4-phenyl C-H), 7.30 (2H, t, J=7.81 Hz, cyclohexyl
4-phenyl C-H), 7.42-7.48 (1H, m, 3-phenyl C-H), 7.49-7.55 (4H, m, 3-phenyl C-H), 7.63 (1H, d, J=8.79
Hz, indole C7-H), 7.71 (1H, dd, J=8.79, 0.98 Hz, indole C6-H), 7.95 (1H, s, indole C4-H), 9.61 (1H, s,
CONH), 12.37 (1H, s, indole NH). Anal. Calcd. for C27H26N4O3S (486.585): C, 66.65; H, 5.39; N, 11.51;
S, 6.59. Found: C, 66.38; H, 5.57; N, 11.88; S, 8.94.
3.1.23. 2-{[2-(4-tert-buthylcyclohexylidene)hydrazinyl]carbonyl}-3-phenyl-1H-indole-5-sulfonamide (22)
White powder (55%); mp 284.9–285.2 ◦C; IR(KBr):
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 0.83 (9H, s, 3 x CH3), 1.10-1.23 (3H, m, -cyclohexyl C-H),
1.45 (1H, td, J=14.15, 4.88 Hz, -cyclohexyl C-H), 1.64 (1H, d, J=10.74 Hz, -cyclohexyl C-H), 1.80-1.89
υ 3358, 3284, 3161 (NH), 1662 (C=O); 1342, 1143
δ

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(2H, m, -cyclohexyl C-H), 2.13 (1H, t, J=10.74 Hz, -cyclohexyl C-H), 2.36 (1H, d, J=12.69 Hz, -cyclohexyl
C-H), 7.16 (2H, s, SO2NH2), 7.47-7.56 (5H, m, 3-phenyl C-H), 7.61 (1H, d, J=8.79 Hz, indole C7-H), 7.71
(1H, d, J=8.79 Hz, indole C6-H), 7.94 (1H, s, indole C4-H), 9.49 (1H, s, CONH), 12.36 (1H, s, indole NH).
Anal. Calcd. for C25H30N4O3S (466.595): C, 64.35; H, 6.48; N, 12.01; S, 6.87. Found: C, 64.28; H, 6.35; N,
11.90; S, 6.47.
3.1.24. 2-{[2-(1-Methylpiperidine-4-ylidene)hydrazinyl]carbonyl}-3-phenyl-1H-indole-5-sulfonamide (23)
White powder (65%); mp 250.1–250.3 ◦C; IR(KBr):
(S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 1.71 (2H, br s, piperidine C3/5-H), 2.17 (5H, br s, N-CH3
υ 3352, 3334, 3305 (NH), 1666 (C=O); 1315, 1159
δ
and piperidine C2/6 -H), 2.30 (2H, br s, piperidine C3/5-H), 2.42 (2H, br s, piperidine C2/6 -H), 7.17 (2H,
s, SO2NH2), 7.38-7.54 (5H, m, 3-phenyl C-H), 7.61 (1H, d, J=8.79 Hz, indole C7-H), 7.72 (1H, dd, J=8.78,
1.46 Hz, indole C6-H), 7.96 (1H, s, indole C4-H), 9.66 (1H, s, CONH), 12.39 (1H, s, indole NH). 13CNMR
(proton decoupled, DMSO-d6, 150 MHz)
δ (ppm): 26.42, 33.99 (piperidine C3/5), 45.20 (N-CH3), 53.88,
55.29 (piperidine C2/6), 112.66 (ind. C7), 118.52 (ind. C4), 118.60 (ind. C3), 121.28 (ind. C6), 125.86
(ind. C3a), 127.59 (3-Phenyl C4), 128.93 (3-Phenyl C3/5), 132.93 (ind. C5), 130.19 (3-Phenyl C2/6), 136.36
(3-Phenyl C1), 136.64 (ind. C7a), 157.51 (N=C), 158.57 (CONH). 13C-NMR (HSQC-2D, DMSO-d6, 125
MHz) δ (ppm): 26.40 [piperidine C3/5 - 1.71 (2H, br s, piperidine C3/5-H)], 33.95 [piperidine C3/5- 2.30
(2H, br s, piperidine C3/5-H)], 45.20 [N-CH3 - 2.17 (5H, br s, N-CH3 and piperidine C2/6-H)], 53.87
[piperidine C2/6- 2.17 (5H, br s, N-CH3 and piperidine C2/6 -H)], 55.28 [piperidine C2/6—2.42 (2H, br s,
piperidine C2/6 -H)], 112.63 [ind. C7 - 7.61 (1H, d, J=8.79 Hz, indole C7-H)], 118.55 [ind C4 -7.96 (1H,
s, indole C4-H)], 121.27 [ind. C6 - 7.72 (1H, dd, J=8.78, 1.46 Hz, indole C6-H)], 127.58 [3-phenyl C4
- 7.38–7.54 (5H, m, 3-phenyl C-H)], 128.92 [3-phenyl C3/5 - 7.38–7.54 (5H, m, 3-phenyl C-H)], 130.14
[3-phenyl C2/6 - 7.38–7.54 (5H, m, 3-phenyl C-H)]. Anal. Calcd. for C21H23N5O3S (425.504): C, 59.28;
H, 5.45; N, 16.46; S, 7.54. Found: C, 58.87; H, 5.30; N, 16.20; S, 7.36.
3.1.25. 2-{[2-(1-Benzylpiperidine-4-ylidene)hydrazinyl]carbonyl}-3-phenyl-1H-indole-5-sulfonamide (24)
White powder (70%); mp 251.2–251.4 ◦C; IR(KBr):
1176 (S=O); 1H NMR (DMSO-d6/500 MHz
(ppm): 1.73 (2H, br s, piperidine C-H), 2.26-2.31 (4H, m,
υ 3379, 3352, 3265 (NH), 1685 (C=O); 1311,
δ
piperidine C-H), 2.48 (2H, s, piperidine C-H), 3.37 (2H, s, N-CH2-), 7.16 (2H, s, SO2NH2), 7.25-7.35
(5H, m, benzyl C-H), 7.39-7.45 (1H, m, 3-phenyl C-H), 7.49-7.55 (4H, m, 3-phenyl C-H), 7.61 (1H, d,
J=8.30 Hz, indole C7-H), 7.72 (1H, d, J=8.79 Hz, indole C6-H), 7.96 (1H, s, indole C4-H), 9.67 (1H, s,
CONH), 12.37 (1H, s, indole NH). 13CNMR (proton decoupled, DMSO-d6, 150 MHz)
δ (ppm): 26.42,
33.93 (piperidine C3/5), 51.50, 52.72 (piperidine C2/6), 61.01 (N-CH2-Ph), 112.53 (ind. C7), 118.45 (ind.
C4), 122.49 (ind. C6), 125.76 (ind. C3a), 126. 84, 127.40 (2 x Phenyl C4), 128.08, 128.61 (2 x Phenyl C3/5),
128.82 (ind. C2), 129.38, 130.08 (2 x Phenyl C2/6), 132.90 (ind. C5), 136.30, 136.57 (2 x Phenyl C1), 138.30
(ind. C7a), 157.50 (N=C), 158.50 (CONH). Anal. Calcd. for C27H27N5O3S (501.599): C, 64.65; H, 5.43; N,
13.96; S, 6.39. Found: C, 64.87; H, 5.20; N,14.22; S, 6.16. m/z ESI (positive) 502.63 (M+H)+.
3.2. Enzyme Inhibition Studies
A stopped-flow instrument (SX.18 MV-R Applied Photophysics model) was used for assaying
the CO2 hydration activity of various CA isozymes, as reported by Khalifah [29]. Phenol red (at a
concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm with
10 mM Hepes (pH 7.4) as a buffer and 0.1 M NaClO4 (for constantly maintaining the ionic strength;
this anion was 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 substrates.
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 were done with the assay buffer mentioned above. At least seven different
inhibitor concentrations were used for measuring the inhibition constant. For allowing the complete
formation of the enzyme-inhibitor adduct, the inhibitor and the enzyme were pre-incubated for 15 min.
IC50 values were obtained from dose response curves working at seven different concentrations of

Int. J. Mol. Sci. 2019, 20, 2354
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the test compound (from 0.1 nM to 50 mM) by fitting the curves using PRISM (www.graphpad.com)
and non-linear least squares methods, the obtained values representing the mean of at least three
different determinations. The inhibition constants (
Cheng-Prusoff equation as follows: I=IC50/(1+[S]/
which the measurement was carried out, and M represents the concentration of the substrate at which
the enzyme activity was at half maximal. As reported earlier, all enzymes used were recombinant and
produced in Escherichia Coli. [30 33]. The concentrations of enzymes used in the assay were: hCA I,
K
I) were derived from the IC50 values by using the
K
KM) where [S] represents the CO2 concentration at
K
–
11.9 nM; hCA II, 7.7 nM; hCA IX, 9.1 nM, and hCA XII, 11.5 nM.
3.3. Molecular Modeling Studies
The three-dimensional structures of all ligands were prepared in their lowest energy conformation
using the MOE software package (v2018.0101, Chemical Computing Group, Inc, Montreal, Canada).
The sulfonamide nitrogen atoms were assigned a negative charge (R-SO2NH-), and the ligands were
energy minimized (MMFF94x force field).
All protein structures were obtained from the RCSB protein databank: hCA I in complex with
topiramate (pdb: 3lxe, 1.90 Å), hCA II in complex with water (pdb: 4e3d, 1.60 Å), hCA IX in complex
with acetazolamide (pdb: 3iai; 2.20 Å), and hCA XII in complex with acetazolamide (pdb: 1jd0;
1.50 Å). The protein atoms and the active site zinc ions were retained, and all other atoms were
omitted. The remaining structure was protonated using the protonate 3D functionality of MOE,
and subsequently, the obtained structure was energy-minimized (AMBER14:EHT) [34]. Finally, the
obtained protein models were superposed on the hCA I structure using the backbone Cα-atoms and all
Zn2+-ions, zinc-binding histidines, and the overall backbone atoms superposed well [root-mean-square
deviation (RMSD) value: 1.281 Å].
Docking calculations were performed using the FlexX docking tool (v2.3.2; BioSolveIT GmbH,
St. Augustin, Germany) within MOE. The binding pocket was defined as all residues within 6.5 Å of
the reference ligand, acetazolamide. The sulfonamide tail of the ligands was forced to adopt a similar
orientation and interactions to the Zn2+-ion as observed for acetazolamide using a pharmacophore
model. All ligands were docked fifty times, and the best scoring three poses were subjected to refinement
calculations. To this end, the ligand and the binding pocket residues were energy minimized and
rescored using a GBVI/WSA force field [35].
4. Conclusions
A total of 21 new indole-based sulfonamide derivatives were synthesized and tested against
the tumor-associated hCA IX and XII and the widespread off-targets, hCA I and II. The compounds
showed potent inhibition in the lower nanomolar range and at least ~six-fold selectivity for hCA IX
and XII over hCA I and II. Molecular modeling studies were subsequently performed to gather insight
in the possible binding modes between the newly developed CAIs and their respective target hCAs.
This information may result in more potent and more selective CAIs for hCA IX and XII.
Author Contributions: Conceptualization, Ö.G.A. and C.T.S.; Synthesis, K.D.Y., N.M.A. and Ö.G.A., Molecular
modeling, A.A. and K.D.Y.; Enzyme inhibition assays: S.B., C.T.S. Writing—Original Draft Preparation, K.D.Y.,
A.A. Ö.G.A.; Writing—Review & Editing, A.A. and Ö.G.A.; Supervision, Ö.G.A.; Project Administration, Ö.G.A.;
Funding Acquisition, Ö.G.A.
Funding: This research was funding by the Istanbul University Scientific Research Projects Department under
project numbers TSA-2019-30535.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

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Abbreviations
CA
Carbonic Anhydrases
hCA
CAI
Human Carbonic Anhydrases
Carbonic Anhydrase Inhibitor
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