Novel sulfonamide-containing 2-indolinones that selectively inhibit tumor-associated alpha carbonic anhydrases

Article abstract of DOI:10.1016/j.bmc.2017.05.029

Human carbonic anhydrases IX and XII are upregulated in many tumors and form a novel target for new generation anticancer drugs. Here we report the synthesis of novel 2-indolinone derivatives with the sulfonamide group as a zinc binding moiety. Enzyme inh

Full text of DOI:10.1016/j.bmc.2017.05.029

Accepted Manuscript
Novel sulfonamide-containing 2-indolinones that selectively inhibit tumor-as-
sociated alpha carbonic anhydrases
Nilgün Karalı, Atilla Akdemir, Füsun Göktaş, Pınar Eraslan Elma, Andrea
Angeli, Merih Kızılırmak, Claudiu T. Supuran
PII:
S0968-0896(17)30572-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.029
BMC 13747
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
29 March 2017
4 May 2017
5 May 2017
Please cite this article as: Karalı, N., Akdemir, A., Göktaş, F., Eraslan Elma, P., Angeli, A., Kızılırmak, M., Supuran,
C.T., Novel sulfonamide-containing 2-indolinones that selectively inhibit tumor-associated alpha carbonic
anhydrases, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.05.029
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Novel sulfonamide-containing 2-indolinones
that selectively inhibit tumor-associated
alpha carbonic anhydrases
Nilgün Karalı, Atilla Akdemir*, Füsun Göktaş, Pınar Eraslan Elma, Andrea Angeli, Merih Kızılırmak and
Claudiu T. Supuran*

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Novel sulfonamide-containing 2-indolinones that selectively inhibit tumor-associated
alpha carbonic anhydrases
Nilgün Karalıa, Atilla Akdemirb*, Füsun Göktaşa, Pınar Eraslan Elmaa, Andrea Angelic, Merih Kızılırmaka,
and Claudiu T. Supuranc*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University, Istanbul, 34116, Turkey
b Computer-Aided Drug Discovery Laboratory, Department of Pharmacology, Faculty of Pharmacy, Bezmialem Vakif University, Istanbul, 34093, Turkey
c Department of NEUROFARBA, Sezione di Scienze Farmaceutiche Universita degli Studi di Firenze, Sesto Fiorentino (Florence), 50019, Italy
* Corresponding authors: AA: Email: aakdemir@bezmialem.edu.tr, Tel: +90-212-5232288/3142; CTS: Email: claudiu.supuran@unifi.it, Tel: +39-055-
4573729/3005
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Human carbonic anhydrases IX and XII are upregulated in many tumors and form a novel target
for new generation anticancer drugs. Here we report the synthesis of novel 2-indolinone
derivatives with the sulfonamide group as a zinc binding moiety. Enzyme inhibition assays
confirmed that the compounds showed selectivity against hCA IX and XII over the widely
distributed off-targets hCA I and II. Molecular modelling studies were performed to suggest
modes of binding for these compounds.
Available online
Keywords:
carbonic anhydrase
isatin
2009 Elsevier Ltd. All rights reserved.
2-indolinone
docking
1. Introduction
analysis of active compounds revealed that 1H-indole-2,3-dione
3-thiosemicarbazone moiety, aromatic/ hydrophobic features at
Carbonic anhydrases are enzymes that are widely distributed
throughout almost all forms of live. They contain a zinc ion in
their active site and they catalyze the hydration of carbon dioxide
to bicarbonate, which is both a simple and at the same time a
physiological important biochemical reaction.1-6 It not only
influences the acid/base balance in tissues and cells, but also is
involved in carbon fixation and control of the bicarbonate levels.
The human carbonic anhydrase isozymes IX and XII (hCA IX
and XII) are upregulated in many tumors and may form a novel
target for new generation anticancer drugs. hCA I and II are
widely distributed throughout the human body and interference
with these enzymes may cause side effects. As such, carbonic
anhydrase inhibitors (CAIs) which show selectivity for hCA IX
and/or hCA XII over hCA I and II are awaited. Here we focus on
a novel class of 2-indolinone containing sulfonamides as putative
CAIs of hCA IX/XII.
the N4 position of the thiosemicarbazone and introduction of
electron-withdrawing groups to 5 and/or 7 positions of 1H-
indole-2,3-dione were essential for anticancer activity.15-17
Additional studies showed that N4-phenyl substituted
thiosemicarbazone derivatives were significantly more active
than N4-alkyl and N4-cycloalkyl thiosemicarbazone derivatives.8,9
In the current study, we have synthesized novel 1H-indole-
2,3-dione 3-(N-phenylthiosemicarbazones) derivatives that all
contained a sulfonamide group as zinc binding moiety. These
compounds were tested in enzyme inhibition assays of the
intended target enzymes hCA IX and XII and the off-targets hCA
I and II. The compounds showed selectivity for hCA IX/XII over
hCA I/II. Molecular modelling studies were performed to gain
more insight into the possible binding interactions of these novel
class of CAIs.
1H-Indole-2,3-dione and its derivatives have a broad spectrum
of biological properties including anticancer, antiviral and
antimicrobial activities. There are several reports on anticancer
2. Results and discussion
activities
of
1H-indole-2,3-dione
3-thiosemicarbazone
2.1. Chemistry
derivatives.7-10 On the other hand, 2-indolinone derivatives
carrying benzenesulfonamide moiety have been reported as
selective carbonic anhydrase inhibitors.4,11-14 Pharmacophore
In this study, 4-isothiocyanatobenzenesulfonamide 1 was
prepared by reacting of 4-aminobenzenesulfonamide with

thiophosgene in water containing concentrated hydrochloric
acid.18 Hydrazine hydrate was reacted with 1 in ethanol to give
N-(4-sulfamoylphenyl)thiosemicarbazide 2. A series of 1H-
indole-2,3-dione 3-[N-(4-sulfamoylphenyl)thiosemicarbazones]
4a-m were synthesized by reacting 2 with 1H-indole-2,3-dione
3a-m in ethanol containing a catalytic amount of sulfuric
acid.7,19,20 The structures of 4a-m were confirmed by analytical
and spectral (IR, 1H NMR, 13C-DEPT, HSQC-2D, HMBC-2D
and LCMS-ESI) data (Scheme 1).
2.2. Carbonic anhydrase enzyme inhibition assays
The 13 ligands have been tested against the two tumor-
associated membrane-bound hCA IX and XII and the widely
distributed cytosolic off-targets hCA I and II (Table 1). The
compounds show KI values in the lower nanomolar range for
hCA XII (1.2 – 87.3 nM) and hCA IX (13.3 – 66.3 nM).
Table 1. CA enzyme inhibition data (KI, nM) of compounds 4
and acetazolamide (AAZ).
Compound
R
hCA I
1190
744
hCA II
hCA IX
66.3
62.3
45.7
38.8
54.2
64.8
16.8
19.5
34.4
20.8
13.3
21.3
58.1
25.0
hCA XII
4a
4b
4c
H
936
6.9
S
C N
H2N
5-CH3
5-OCF3
5-F
735
5.7
Cl
Cl
i
481
420
6.0
S
C
+
4d
4e
558
551
4.5
SO2NH2
SO2NH2
1
5-Cl
650
616
5.8
ii
4f
5-Br
882
851
6.1
H2N NH2
NH2
HN
4g
4h
4i
5-I
624.2
68.8
901
399.4
32.0
897
5.9
NH
C
5-SO3Na
5-NO2
7-F
4.1
S
1.2
SO2NH2
2
4j
57.6
76.1
554.4
698
26.9
425.6
782.1
687
45.5
87.3
82.3
6.3
O
4k
4l
7-Cl
5,7-diCl
5,7-diBr
---
iii
O
R
N
H
4m
AAZ
250
12.1
5.7
3a-m
R
The measured KI values for hCA IX are higher compared to
hCA XII (still in lower nanomolar region), but there is only
approximately 5-fold difference between the highest and lowest
measured KI values, resulting in a flat SAR (Table 1).
SO2NH2
NH
N
C
NH
NH
S
O
The measured inhibition constant for hCA XII are lower for
most compounds, except for 4j, 4k and 4l. These three
compounds have KI values higher than 45 nM, while all other
compounds have KI values lower than 7 nM. The lowest KI value
has been measured for 4i (1.2 nM). With the exception of
compounds 4j, 4k and 4l only approximately 6-fold difference in
KI values has been observed, which makes the structure-activity
relationships (SAR) data rather flat and difficult to interpret.
Compounds 4j, 4k and 4l have either a fluorine or chlorine
substituent at position 7 (in addition also 5-position for
compound 4l). However, compound 4m has bromine substituents
at the 5- and 7-positions, which is well tolerated. In addition, it
seems that negatively charged substituents at the 5-position (such
as 4i and 4h) show the lowest KI values.
4a-m
R=
H, 5-CH3,
5-F3CO,
5-F, 5-CI,
5-Br, 5-I
,
5
-SO3Na,
5-NO , 7-F, 7-Cl,
5,7-diCl, 5
,7-diBr
2
Scheme 1. Synthesis of 4a-m. Reagents and conditions: i) H2O,
HCI, stirred ii) EtOH, stirred, cooled ii) EtOH, H2SO4, reflux
IR spectra of 4a-m showed absorption bands in the 3394-3115
cm-1 region resulting from the NH stretching of the lactam,
thioamide and sulfonamide functions. The lactam C=O and
thioamide C=S stretching were observed in the 1712-1674 and
1224-1204 cm-1 regions, respectively. The sulfonamide S=O
stretching showed two separate absorptions at 1343-1311 and
1161-1138 cm-1 regions.21-23 1H NMR spectra of 4a-m displayed
the sulfonamide NH protons (δ 7.36-7.60 ppm), the NH protons
of the thiosemicarbazone moiety (δ 10.93-11.22 and 12.65-12.92
ppm) and the indole NH proton (δ 11.15-11.86 ppm) as four
separate singlets. HSQC-2D spectra of 4a and HMBC-2D spectra
of 4d showed the C=O (δ 163.41 and 163.49 ppm) and the C=S
(δ 177.04 and 177.09 ppm) peaks which verified the
thiosemicarbazone structure.22,24-26 In the mass spectra of 4a, 4d
and 4m (M-H)- peaks were observed which confirmed their
molecular weights.
2.3. Molecular modelling studies
All KI values of the tested compounds are in the lower
nanomolar range (< 90 nM) for hCA IX and XII, with the lowest
measured KI value for compound 4i (hCA XII; Table 1). The
docked pose for this compound suggests that the nitro (at 5-
position) and carbonyl groups chains of His64 and Lys67 of hCA
XII, respectively (Figure 1). It should be noted that the nitro
group is positioned close to the cationic nitrogen atom of Lys170
(distance < 3.0 Å) and that electrostatic interactions are possible.
The sulfonamide tail interacts with the active site Zn2+ with its
nitrogen and oxygen atoms to form distorted trigonal bipyramidal
geometry. Compound 4h has an identical binding pose and the

3. Experimental protocols
sulfonate group forms hydrogen bonds with both His64 and
Lys170.
3.1. Chemistry
All docked compounds can adopt this docked pose in the hCA
XII active site. The additional hydrogen bonds and electrostatic
interactions formed by the nitro and sulfonate substituents at the
5-position are expected to be the cause for the lower KI values of
4i and 4h.
All reagents and solvents were purchased from Sigma-
Aldrich (Darmstadt, Germany) or Meck (Germany) used as
received. Compound 3c was obtained from (Sigma-Aldrich,
390674). Melting points were estimated with a Buchi 540
melting point apparatus in open capillaries and are uncorrected.
Elemental analyses were performed on a Thermo Finnigan Flash
EA 1112 elemental analyzer. IR spectra were recorded on KBr
1
discs, using a Perkin-Elmer Model 1600 FT-IR spectrometer. H
NMR, HSQC-2D and HMBC-2D spectra were obtained on
Varian UNITY INOVA 500 spectrophotometer using DMSO-d6.
Mass spectra were determined on an AGILENT 1100 MSD
instrument.
3.1.1. The synthesis of 4-
isothiocyanatobenzenesulfonamide (1)
4-Aminobenzenesulfonamide (3 mmol) was dissolved in 50
mL of water containing 12 mL of concentrated hydrochloric acid.
To the solution, thiophosgene (3 mmol) was added in one
portion. Stirring was begun immediately and continued until all
of the red color of thiophosgene had disappeared and the product
appeared as a white crystalline precipitate. The precipitate was
filtered, washed thoroughly with water, and recrystallized from
acetone-water.
3.1.2. The synthesis of N-(4-sulfamoylphenyl)
thiosemicarbazide (2)
Figure 1. The docked pose of compound 4i in the active site of hCA XII.
To a solution of hydrazine hydrate (5 mmol) in ethanol (10
mL), a suspension of 4-isothiocyanatobenzenesulfonamide 1 (5
mmol) in ethanol (10 mL) was added dropwise with vigorous
stirring and cooling in an ice bath. The mixture was allowed to
stand overnight. The crystals formed were recrystallized from
ethanol.
3.1.3. The synthesis of 1H-indole-2,3-dione 3-[N-
(4-sulfamoylphenyl)thiosemicarbazones] (4a-m)
N-(4-sulfamoylphenyl)hydrazinecarbothioamide (2) (3.5
mmol) was added to a solution of 1H-indole-2,3-diones 3a-m
(3.5 mmol) in ethanol (20 mL). After addition of a drop of
concentrated sulfuric acid, the mixture was refluxed on a water
bath for 4 h. The product formed after cooling was filtered and
washed with ethanol or recrystallized from ethanol.
Figure 2. The docked poses of compounds 4i (left panel; turquoise) and 4k
(right panel; magenta) in the active site of hCA IX.
The hCA IX active site shows some minor but important
differences compared to the hCA XII site. Gln67 (hCA IX) is
present in the active site instead of Lys67 (hCA XII) resulting in
a loss of the hydrogen bond with the carbonyl of the 2-indolinone
ring. Additionally, a negatively charged Glu170 is present in
hCA IX instead of the cationic Lys170 in hCA XII. This may
interfere with the negatively charged nitro or sulfonate
substituents at the 5-position of the 2-indolinone ring. As such,
the compounds adopt two different docked poses in the hCA IX
active site (Figure 2). Both poses show hydrophobic interactions
between their phenyl groups and the side chain of Leu198 and
hydrogen bonds between either their carbonyl group or sulfur
atoms and the side chain of Gln92. The nitro substituent at the 5-
position of compound 4i forms an additional hydrogen bond with
the backbone of Val131. The chlorine substituent of the 7-
position of compound 4k interact with the side chain of Asp132.
The other compounds can adopt similar poses as shown in Figure
2.
3.1.4. 1H-indole-2,3-dione 3-[N-(4-
sulfamoylphenyl)thiosemicarbazones] (4a)27
Yellow powder (90%): mp 268oC; IR (KBr): υ 3374, 3274,
1
3182 (NH), 1674 (C=O), 1337, 1154 (S=O), 1207 (C=S). H
NMR (DMSO-d6/ 500 MHz) ppm: 6.94 (1H, d, J: 7.80 Hz, ind.
C7-H), 7.10 (1H, dt, J: 7.80, 7.32 Hz, ind. C5-H), 7.36 (2H, s,
SO2NH2, D2O exch.), 7.37 (1H, t, J: 7.80 Hz, ind. C6-H), 7.76
(1H, d, J: 7.32 Hz, ind. C4-H), 7.85 (4H, s, phen.), 10.94 (1H, s,
N4-H, D2O exch.), 11.25 (1H, s, ind. NH, D2O exch.), 12.89 (1H,
s, N2-H, D2O exch.). HSQC-2D (DMSO-d6/125 MHz): 111.87
(ind. C7), 120.49 (ind. C3a), 122.17 (ind. C4), 123.11 (ind. C6),
125.98 (phen. C2,6), 126.67 (phen. C3,5), 132.34 (ind. C5),
133.55 (ind. C3), 141.79 (ind. C7a), 142.14 (phen. C4), 143.34
(phen. C1), 163.41 (ind. C2), 177.09 (C=S). LCMS-ESI (-): m/z
(%) 374 (M-H-, 100). Anal. Calcd for C15H13N5O3S2: C, 47.99;
H, 3.49; N, 18.65 Found: C, 47.68; H, 3.56; N, 18.17.

phen.), 11.00 (1H, s, N4-H), 11.35 (1H, s, ind. NH.), 12.70 (1H,
s, N2-H). Anal. Calcd for C15H12BrN5O3S2: C, 39.65; H, 2.66; N,
15.41 Found: C, 39.72; H, 2.66; N, 15.09.
3.1.5. 5-Methyl-1H-indole-2,3-dione 3-[N-(4-
sulfamoylphenyl)thiosemicarbazones] (4b)
Yellow powder (93%): mp 258-260oC; IR (KBr): υ 3296,
3269, 3173 (NH), 1685 (C=O), 1329, 1153 (S=O), 1212 (C=S).
1H NMR (DMSO-d6/ 500 MHz) ppm: 2.30 (3H, s, CH3), 6.83
(1H, d, J: 8.33 Hz, ind. C7-H), 7.18 (1H, d, J: 8.33 Hz, ind. C6-
H), 7.36 (2H, s, SO2NH2), 7.59 (1H, s, ind. C4-H), 7.87 (4H, s,
phen.), 10.93 (1H, s, N4-H), 11.15 (1H, s, ind. NH), 12.88 (1H, s,
N2-H). Anal. Calcd for C16H15N5O3S2: C, 49.34; H, 3.88; N,
17.98 Found: C, 49.12; H, 3.89; N, 17.85.
3.1.10. 5-Iodo-1H-indole-2,3-dione 3-[N-(4-
sulfamoylphenyl)thiosemicarbazones] (4g)
2 8 , 29
Yellow powder (73%): mp 286-288oC; IR (KBr): υ 3307,
3247, 3176 (NH), 1683 (C=O), 1327, 1151 (S=O), 1205 (C=S).
1H NMR (DMSO-d6/ 500 MHz) ppm: 6.99 (1H, d, J: 8.30 Hz,
ind. C7-H), 7.60 (2H, s, SO2NH2), 7.88 (1H, dd, J: 8.30, 1.20 Hz,
ind. C6-H), 8.34 (1H, d, J: 1.20 Hz, ind. C4-H), 8.05 (4H, s,
phen.), 11.22 (1H, s, N4-H), 11.56 (1H, s, ind. NH.), 12.92 (1H,
s, N2-H); Anal. Calcd for C15H12IN5O3S2: C, 35.94; H, 2.41; N,
13.97 Found: C, 35.81; H, 2.62; N, 13.71.
3.1.6. 5-Trifluoromethoxy-1H-indole-2,3-dione 3-
[N-(4-sulfamoylphenyl)thiosemicarbazones] (4c)
Light yellow powder (90%): mp 246-248oC; IR (KBr): υ
3339, 3237, 3115 (NH), 1693 (C=O), 1326, 1153 (S=O), 1204
(C=S). 1H NMR (DMSO-d6/ 500 MHz) ppm: 7.03 (1H, d, J: 8.32
Hz, ind. C7-H), 7.35 (1H, dd, J: 8.32, 1.60 Hz, ind. C6-H), 7.37
(2H, s, SO2NH2, D2O exch.), 7.78 (1H, d, J: 1.60, Hz, ind. C4-H),
7.84 (2H, s, phen. C2-H, C6-H), 7.85 (2H, s, phen. C3-H, C5-H),
11.01 (1H, s, N4-H, D2O exch.), 11.41 (1H, s, ind. NH, D2O
exch.), 12.75 (1H, s, N2-H, D2O exch.). Anal. Calcd for
C16H12F3N5O4S2: C, 41.83; H, 2.63; N, 15.24 Found: C, 41.49; H,
2.52; N, 15.16.
3.1.11. 5-Nitro-1H-indole-2,3-dione 3-[N-(4-
sulfamoylphenyl)thiosemicarbazones] (4h)
Dark yellow powder (85%): mp 265-267oC; IR (KBr): υ 3546,
3329, 3256 (NH), 1692 (C=O), 1343, 1155 (S=O), 1213 (C=S).
1H NMR (DMSO-d6/ 500 MHz) ppm: 1.04 (3H, t, J: 6.83 Hz,
CH3CH2OH), 3.43 (2H, q, J: 6.83 Hz, CH3CH2OH), 4.31 (1H,
br.s, CH3CH2OH), 7.13 (1H, d, J: 8.78 Hz, ind. C7-H), 7.37 (2H,
s, SO2NH2), 8.28 (1H, dd, J: 8.78, 2.44 Hz, ind. C6-H), 8.67 (1H,
d, J: 2.44 Hz, ind. C4-H), 7.84 (2H, s, phen. C2-H, C6-H) , 7.86
(2H, s, phen. C3-H, C5-H), 11.19 (1H, s, N4-H), 11.86 (1H, s,
ind. NH.), 12.65 (1H, s, N2-H); Anal. Calcd for
C15H12N6O5S2.1/2C2H5OH: C, 43.20; H, 3.37; N, 18.91 Found: C,
42.96; H, 3.38; N, 18.92.
3.1.7. 5-Fluoro-1H-indole-2,3-dione 3-[N-(4-
sulfamoylphenyl)thiosemicarbazones] (4d)
Red powder (92%): mp 260-262oC; IR (KBr): υ 3338, 3236,
1
3115 (NH), 1693 (C=O), 1326, 1154 (S=O), 1205 (C=S). H
NMR (DMSO-d6/ 500 MHz) ppm: 6.94 (1H, dd, J: 8.30, 3.91 Hz,
ind. C7-H), 7.20 (1H, dd, J: 8.78, 2.44 Hz, ind. C6-H), 7.36 (2H,
s, SO2NH2), 7.62 (1H, dd, J: 8.30, 2.44 Hz, ind. C4-H), 7.85 (4H,
s, phen.), 10.96 (1H, s, N4-H), 11.25 (1H, s, ind. NH), 12.77 (1H,
s, N2-H); HMBC-2D (DMSO-d6/ 125 MHz): 109.06 (d, J: 25.88
Hz, ind. C4), 112.96 (d, J: 8.14 Hz, ind. C7), 118.56 (d, J: 24.44
Hz, ind. C6), 121.92 (d, J: 9.58 Hz, ind. C3a), 125.98 (phen.
C2,6), 126.73 (phen. C3,5), 132.91 (d, J: 2.87 Hz, ind. C3),
139.60 (d, J: 1.91 Hz, ind. C7a), 141.94 (phen. C4), 141.95
(phen. C1), 158.98 (d, J: 237.71, ind. C5), 163.49 (ind. C2),
177.04 (C=S). LCMS-ESI (-): m/z (%) 392 (M-H-, 100). Anal.
Calcd for C15H12FN5O3S2: C, 45.79; H, 3.07; N, 17.80 Found: C,
45.46; H, 3.26; N, 17.33.
3.1.12. Sodium 1H-indole-2,3-dione 3-[N-(4-
sulfamoylphenyl)thiosemicarbazones] - 5-sulfonate
(4i)2 8 ,2 9
Dark yellow powder (66%): mp >300oC; IR (KBr): υ 3271,
1
3234 (NH), 1712 (C=O), 1313, 1153 (S=O), 1220 (C=S). H
NMR (DMSO-d6/ 500 MHz) ppm: 6.89 (1H, d, J: 8.40 Hz, ind.
C7-H), 7.38 (2H, s, SO2NH2), 7.65 (1H, dd, J: 8.00, 1.60 Hz, ind.
C6-H), 7.84 (4H, s, phen.), 8.16 (1H, d, J: 1.60 Hz, ind. C4-H),
11.20 (1H, s, N4-H), 11.33 (1H, s, ind. NH.), 12.86 (1H, s, N2-
H); Anal. Calcd for C15H12N5NaO6S3.4H2O: C, 32.78; H, 3.85; N,
12.74 Found: C, 32.86; H, 3.69; N, 12.78.
3.1.13. 7-Fluoro-1H-indole-2,3-dione 3-[N-(4-
2 8 ,2 9
sulfamoylphenyl)thiosemicarbazones] (4j)
3.1.8. 5-Chloro-1H-indole-2,3-dione 3-[N-(4-
Yellow powder (86%): mp 271-273oC; IR (KBr): υ 3394,
3277, 3136 (NH), 1681 (C=O), 1338, 1149 (S=O), 1220 (C=S).
1H NMR (DMSO-d6/ 500 MHz) ppm: 7.14 (1H, dd (t), J: 7.80,
4.88 Hz, ind. C5-H), 7.31 (1H, dd, J: 8.29, 1.95 Hz, ind. C6-H),
7.39 (2H, s, SO2NH2, D2O exch.), 7.63 (1H, d, J: 7.32 Hz, ind.
C4-H), 7.85 (4H, s, phen.), 11.04 (1H, s, N4-H, D2O exch.),
11.81 (1H, s, ind. NH, D2O exch.), 12.86 (1H, s, N2-H, D2O
exch.); Anal. Calcd for C15H12FN5O3S2: C, 45.79; H, 3.07; N,
17.80 Found: C, 45.64; H, 2.89; N, 17.52.
sulfamoylphenyl)thiosemicarbazones] (4e)
Orange powder (93%): mp 266-268oC; IR (KBr): υ 3323,
3291, 3165 (NH), 1691 (C=O), 1311, 1161 (S=O), 1204 (C=S).
1H NMR (DMSO-d6/ 500 MHz) ppm: 6.95 (1H, d, J: 8.30 Hz,
ind. C7-H), 7.40 (1H, dd, J: 8.30, 2.44 Hz, ind. C6-H), 7.84 (1H,
d, J: 2.44 Hz, C4-H), 7.36 (2H, s, SO2NH2), 7.85 (4H, s, phen.),
10.99 (1H, s, N4-H), 11.34 (1H, s, ind. NH), 12.71 (1H, s, N2-
H). Anal. Calcd for C15H12ClN5O3S2: C, 43.96; H, 2.95 N, 17.09
Found: C, 43.64; H, 2.86; N, 17.09.
3.1.14. 7-Chloro-1H-indole-2,3-dione 3-[N-(4-
sulfamoylphenyl)thiosemicarbazones] (4k)
3.1.9. 5-Bromo-1H-indole-2,3-dione 3-[N-(4-
sulfamoylphenyl)thiosemicarbazones] (4f)
2 8 , 29
Yellow powder (87%): mp 288-289oC; IR (KBr): υ 3331,
3271, 3130 (NH), 1693 (C=O), 1334, 1138 (S=O), 1224 (C=S).
1H NMR (DMSO-d6/ 500 MHz) ppm: 7.15 (1H, t, J: 7.80 Hz,
ind. C5-H), 7.46 (1H, d, J: 7.80 Hz, ind. C6-H), 7.39 (2H, s,
SO2NH2, D2O exch.), 7.75 (1H, d, J: 7.32 Hz, ind. C4-H), 7.85
Dark yellow powder (96%): mp 254-255oC; IR (KBr): υ 3306,
3245, 3172 (NH), 1687 (C=O), 1326, 1151 (S=O), 1204 (C=S).
1H NMR (DMSO-d6/ 500 MHz) ppm: 6.90 (1H, d, J: 8.33, Hz,
ind. C7-H), 7.58 (1H, dd, J: 8.33, 2.25 Hz, ind. C6-H), 7.36 (2H,
s, SO2NH2), 7.99 (1H, d, J: 2.25, Hz, ind. C4-H), 7.85 (4H, s,

(4H, s, phen.), 11.04 (1H, s, N4-H, D2O exch.), 11.79 (1H, s, ind.
NH, D2O exch.), 12.83 (1H, s, N2-H, D2O exch.); Anal. Calcd for
C15H12ClN5O3S2: C, 43.96; H, 2.95; N, 17.09 Found: C, 44.08; H,
3.34; N, 16.93.
3.3.2. Preparation of protein structures
All protein structures were obtained from the RCSB protein
databank: hCA I (pdb: 3lxe, 1.90 Å), hCA II (pdb: 4e3d, 1.60
Å), hCA IX (pdb: 3iai; 2.20 Å) and hCA XII (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 MOE software package and subsequently
the obtained structure was energy-minimized (AMBER12:EHT
force field). 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 (RMSD value: 1.281 Å).
3.1.15. 5,7-Dichloro-1H-indole-2,3-dione 3-[N-(4-
2 8 ,2 9
sulfamoylphenyl)thiosemicarbazones] (4l)
Yellow powder (77%): mp 275-277oC; IR (KBr): υ 3321,
3251, 3136 (NH), 1693 (C=O), 1330, 1153 (S=O), 1222 (C=S).
1H NMR (DMSO-d6/ 500 MHz) ppm: 7.37 (2H, s, SO2NH2), 7.59
(1H, d, J: 1.97 Hz, ind C6-H), 7.85 (3H, br.s, phen. C2-H, C6-H,
ind. C4-H), 7.86 (2H, s, phen. C3-H, C5-H) , 11.04 (1H, s, N4-
H), 11.82 (1H, s, ind. NH), 12.65 (1H, s, N2-H); Anal. Calcd for
C15H11Cl2N5O3S2: C, 40.55; H, 2.50; N, 15.76 Found: C, 40.38;
H, 2.80; N, 15.73.
3.3.3. Docking protocols
The GOLD Suite software package (v5.5, CCDC, Cambridge,
UK) and the ChemScore scoring function were used to dock the
compounds into the hCA structures (50 dockings per ligand). The
binding pocket was defined as all residues within 13 Å of a
3.1.16. 5,7-Dibromo-1H-indole-2,3-dione 3-[N-(4-
sulfamoylphenyl)thiosemicarbazones] (4m)
−17.071, y: 35.081, 43.681; corresponding
centroid (x:
Yellow powder (95%): mp 283oC; IR (KBr): υ 3328, 3255,
approximately to the position of the thiadiazole ring of
acetazolamide in the 1jd0 structure). Position restraints were
applied to the sulfur and nitrogen atoms of the acetazolamide
sulfonamide tail of hCA XII (default settings) and were also
applied to the other three hCA structures due to the low RMSD
value of the superpositions.
1
3215 (NH), 1694 (C=O), 1329, 1151 (S=O), 1205 (C=S). H
NMR (DMSO-d6/ 500 MHz) ppm: 7.37 (2H, s, SO2NH2), 7.80
(1H, d, J: 1.92 Hz, ind C6-H), 7.84 (2H, s, phen. C2-H, C6-H),
7.86 (2H, s, phen. C3-H, C5-H) , 8.01 (1H, d, J:1.92 Hz, ind. C4-
H), 11.04 (1H, s, N4-H), 11.69 (s, 1H, ind. NH.), 12.65 (s, 1H,
N2-H). 13C-NMR (DEPT) (DMSO-d6/125MHz) ppm: 124.00
(ind. C4), 126.17 (phen. C2, C6), 126.75 (phen. C3, C5), 138.00
(ind. C6). LCMS-ESI (-): m/z (%) 530, 532, 534 (M-H-, 43, 100,
52). Anal. Calcd for C15H11Br2N5O3S2: C, 33.79; H, 2.08; N,
13.13 Found: C, 33.85; H, 2.65; N, 13.20.
Acknowledgments
This work was supported by Istanbul University Scientific
Research Projects. Project Number: ÖNAP-36808.
3.2. Carbonic anhydrase enzyme inhibition studies
References and notes
A
stopped-flow
instrument
(SX.18MV-R
Applied
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