Synthesis of 1,4-bis(indolin-1-ylmethyl)benzene derivatives and their structure-activity relationships for the interaction of human carbonic anhydrase isoforms i and II

Article abstract of DOI:10.1016/j.bmc.2012.09.027

Several 1,4-bis(indolin-1-ylmethyl)benzene-based compounds containing substituents such as five, six and seven cyclic derivatives on indeno part (9a-c) were prepared and tested against two members of the pH regulatory enzyme family, carbonic anhydrase (CA

Full text of DOI:10.1016/j.bmc.2012.09.027

Bioorganic & Medicinal Chemistry 21 (2013) 1477–1482
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Bioorganic & Medicinal Chemistry
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Synthesis of 1,4-bis(indolin-1-ylmethyl)benzene derivatives and their
structure–activity relationships for the interaction of human carbonic
anhydrase isoforms I and II
Oktay Talaz a, , Hüseyin Çavdar b, Serdar Durdagi c, Hacer Azak a, Deniz Ekinci d
⇑
a Karamanog˘lu Mehmetbey University, Kamil Özdag˘ Science Faculty, Chemistry Department, 70100 Karaman, Turkey
b Dumlupınar University, Education Faculty, 43100 Kütahya, Turkey
c Institute for Biocomplexity and Informatics, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
d Ondokuz Mayıs University, Faculty of Agriculture, Department of Agricultural Biotechnology, 55139 Samsun, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 10 October 2012
Several 1,4-bis(indolin-1-ylmethyl)benzene-based compounds containing substituents such as five, six
and seven cyclic derivatives on indeno part (9a–c) were prepared and tested against two members of
the pH regulatory enzyme family, carbonic anhydrase (CA). The inhibitory potencies of the compounds
at the human isoforms hCA I and hCA II targets were analyzed and KI values were calculated. KI values
Keywords:
Carbonic anhydrase
Docking
Bisindole
Glaucoma
of compounds for hCA I and hCA II human isozymes were measured in the range of 39.3–42.6
lM and
0.17–0.29 M, respectively. The structurally related compound indole was also tested in order to under-
l
stand the structure–activity relationship. Most of the compounds showed good CA inhibitory efficacy. In
silico docking studies of these derivatives within hCA I and II were also carried out and results are sup-
ported the kinetic assays.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) form a family of metallo-
enzymes that play an essential role in several physiological and
Indole ring containing derivatives continue to receive substan-
tial interest because of their broad biological activity range.1,2 In-
dole moieties occur in many synthetic and natural products,
some of them are physiologically relevant and therapeutically
used.3–5 For example bisindole alkaloids are prevalent subclass of
compounds containing indole ring, such as nortopsentins A–C
(1), arcyriaflavin A (2), hyrtiosin B and psychotrimine (4) have at-
tracted interest of researchers due to their biologicalactivities in
recent years (Fig. 1).6,7
Most of the methods used to form bisindoles have been in-
spired over the years by various synthetic strategies developed
for bisindole ring formation. Two indole ring of 3-subtituted, 2-
substitued, fused benzene ring and N-substituted bisindoles can
be possibly found in fused or opens systems. N-substituted and
C2 substituted bisindoles are not common due to less reactive
N and C2 position compared to C3 position.8 However bisFischer
indolizations have remained elusive to date. In this study we re-
port synthesis of bisphenylhydrazine using Fischer indolization
using several ketones.
pathological processes. There are sixteen CA isoforms identified
in mammals (humans have only 15 isoforms) that differ in their
subcellular localization and catalytic activity.9 Inhibitors or activa-
tors of this metalloenzyme family have several medical applica-
tions. For example treatment of glaucoma, in the management of
several neurological disorders including epilepsy, possibly in the
treatment of Alzheimer’s disease, and several agents show clinical
evaluations as antiobesity or antitumor drugs/diagnostic tools.9–12
So far inhibitory effects of different sulfonamide derivatives, an-
ions, metal ions, phenols and drugs have been investigated against
many CAs.11–15
In a recent study, the antioxidant activities of 5,10-dihydroinde-
no[1,2-b]indole derivatives were reported by one of our groups.16
For this reason, we extend the previous study through investigat-
ing the effects of these molecules on the CA activity, with the goal
to discover new CA inhibitors (CAIs).
2. Results and discussion
2.1. Chemistry
Based on our structure–activity relationship analysis, we syn-
thesized a series of new N-connected bisindole alkoloids as shown
⇑
Corresponding author. Tel.: +90 338 2262431.
E-mail address: otalaz@kmu.edu.tr (O. Talaz).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.027

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O. Talaz et al. / Bioorg. Med. Chem. 21 (2013) 1477–1482
H
N
H
N
O
O
OH
O
R
R
N
O
HO
N
H
HN
NH
N
H
N
H
N
H
1
3
2
NHMe
N
N
H
H
N
MeHN
4
Figure 1.
NH2
BrH2C
CH2Br
:
N
NaH
N
NH2
NH
NH2
:
THF
THF
N2
N
H2N
5
4
7
Scheme 1. Synthesis of 1,4-bis((1-phenylhydrazinyl)methylbenzene (7).
n
N
N
NH2
O
N
G. AcOH
TFA
+
N
H2N
n
7
8a-c
n
9a-c
Scheme 2. Synthesis of compounds 9a–c.
in Scheme 1. NaH base was added to the dissolved phenlhydrazine
mixture contained in THF. Phenylhydrazine base developed anion
2.2. CA inhibition studies
by replacing
a
-NH proton. New developed anion attacked on 1,2-
The 1,4-bis(indolin-1-ylmethyl)benzene derivatives 9a–c and
indole have been tested for the inhibition of two cytosolic ubiqui-
tous isozymes of human origin, that is, hCA I and hCA II (Table 1).
bis(bromomethyl)benzene as nucleophyllic attack and resulted in
the synthesis of 1,4-bis((1-phenylhydrazinyl)methyl)benzene.
After synthesis of bisphenylhydrazine, we first used Fischer ind-
olization for one-pot synthesis of bisindole derivatives. Bisindole
alkaloids 9a–c were synthesized using cyclopentanone, cyclohexa-
none and cycloheptanone Fischer indolization with 1,4-bis((1-
phenylhydrazinyl)methyl)benzene by AcOH/H2SO4 acidic media.
The structures were confirmed by 1H NMR characteristic peaks.
(i.e., CH2 protons observed between 5.19 and 5.27 ppm). We tried
also to synthesize eight-membered ring derivatives using cyclooc-
tanone, however synthesis was not successful. Although we tried
different acidic media and temperature, synthesis of this com-
pound could not be achieved (Scheme 2).
Table 1
Ki Values obtained from regression analysis graphs for hCA I and hCA II in the
presence of different inhibitors concentrations (lM)
Compound
hCA I
hCA II
9a
9b
42.6
39.3
41.7
27.4
0.17
0.25
0.29
32.5
9c
Indolea
a
Ref. 13a.

O. Talaz et al. / Bioorg. Med. Chem. 21 (2013) 1477–1482
1479
Figure 2. Top docking pose of compound 9a at the hCA-II.
(KI-s: 0.17–0.29
was the bulky 1,4-bis(indolin-1-ylmethyl)benzene 9a (KI
l
M) (Table 1). The most potent inhibitor
Table 2
Molecular docking binding scores of the compounds 9a–c, EZA and ZNS within hCA I
and II
0.17 M). ChemScore docking scores of compounds 9a–c
l
show similar results. However GOLD Fitness score of com-
pound 9a is better than other derivatives and supports the
obtained experimental results regarding this compound
(Table 2). Also, hydrophobic group containing compounds
9a–c were much more effective compared to indole.
Obtained docking scores of 9a–c and indole against hCAII
supports in vitro data.
Compound Average
Average
Average
ChemScorefor GOLDFitness
Average
ChemScore for GOLDFitness
hCA-I (kJ/mol) Score for hCA-I hCA-II(kJ/mol) Scorefor
hCA-II
9a
9b
ꢀ33.00
ꢀ33.65
ꢀ33.57
ꢀ34.98
48.56
49.11
48.01
58.15
ꢀ48.63
ꢀ48.66
ꢀ48.95
ꢀ41.50
79.10
65.18
64.32
43.59
9c
Indolea
a
Ref. 13a
2.3. In silico studies
The following should be noted regarding CA inhibitory data of
Table 1:
In this study, a flexible docking methodology was employed
using the GOLD docking algorithm.13,14 The tested derivatives were
docked at the binding site of the hCA I and hCA II targets. Average
GOLD Fitness and ChemScore docking scores of docked inhibitors
at hCA I and hCA II targets and corresponding binding interactions
for the most potent inhibitors were tabulated at Tables 2 and 3,
respectively. (Chemscores results for compounds are also detailed
at the Supplementary data Fig. 1.)
(i) The indole derivatives investigated here against the slow
cytosolic izoform hCA I, showed weak to moderate inhibitory
properties. Thus, derivatives 9a and 9c exhibited moderate
inhibition of this isoform, with KI-s in the range of 41.7 and
42.6
effective inhibitors against hCA I, with KI-s in the range of
27.4–39.3 M (Fig. 2). These results demonstrate the contri-
lM (Table 1). The compounds 9b and indole were more
We have performed docking studies of compounds 9a–c within
the hCA I and II active sites, using as templates hCA I and II adducts
for which the X-ray crystal structures have been reported in com-
l
bution of the hydrophobic groups to the inhibition efficacy.
Interestingly, hydrophobic indole group containing com-
pounds 9a–c were as effective as indole. This trend also
shows the efficacy of indole moiety. The most potent deriva-
tives were the compounds 9a–c against hCA II and this data is
supported by the in silico docking data (Table 2). As these
compounds do not possess any of the zinc-anchoring groups
present in known CAIs, presumably such compounds may
bind in the coumarin/activator binding site.16
plexes with activators (e.g., hCA I–L
-His, PDB code 2FW4)15 or
inhibitors (the hCA II–phenol adduct PDB code 2HNC).12,13 Top
docking poses derived for the best inhibitor 9a (against hCA II). It
may be observed that as for phenol5 or hydroxycinnamic acids/
lacosamide20—for which the X-ray crystal struyctures in adduct
with hCA II have been reported—the indoles investigated here do
not coordinate to the metal ion but are bound more towards the
exit of the cavity, interacting eventually with the zinc-coordinated
water molecule. This is a new binding mode within the CA active
site, which was less investigated to date.
(ii) Indole acted as weak inhibitor against the ubiquitous
and dominant rapid cytosolic isozyme hCA II (Table 1).
However, 9a–c derivatives acted as strong hCA II inhibitors
Table 3
Binding interactions for top docking poses of 9a–c within hCAI and II
Compound hCA-I
hCA-II
9a
Ala121, Leu131, Ala135, Leu198, Trp5, Pro201, His200,
Thr199, His94
Phe70, Ile91, Glu69, His119, Val135, Trp123, Gln92, Leu141, Val135, Val121, His122, Leu198,
Pro202, Trp123
9b
9c
Tyr20, Phe91, Leu141, Pro201, His200, Phe95, His96, His94 Glu69, Ile91, Val121, Pro202, Val135, Phe131, Leu198, Gln92
His94, Phe91, Leu141, Pro201, His64 Ln92, Phe131, Ile91, Trp123, Val121, Val135, Pro202, Ile91, Glu69, Phe70, Leu141, Leu57

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O. Talaz et al. / Bioorg. Med. Chem. 21 (2013) 1477–1482
4.2.4. 1,4-bis((2,3-dihydro-1H-Carbazol-9(2H)-
3. Conclusions
yl)methyl)benzene (9b)
Light yellow crystals (2.4 g, 89%) from EtOAc/Hexane; mp 160–
161 °C; 1H NMR (400 MHz, CDCl3) d (ppm): d 7.53–7.49 (m, ArH,
2H), 7.21–7.07 (m, ArH, 6H), 6.80 (s, ArH, 4H), 5.20 (s, ArH, 4H),
2.77–2.75 (m, CH2, 4H), 2.61–2.59 (m, CH2, 4H), 1.90–1.89 (m,
CH2, 8H). 13C NMR (50 MHz, CDCl3) d (ppm): 139.33, 139.56
137.42, 129.55, 122.74, 120.85, 118.78, 111.97, 110.88, 47.90,
25.24, 25.19, 24.15, 23.10. IR (KBr, cmꢀ1): 3053.2, 2924.4, 2851.4,
1767.8, 1605.6, 1465.7, 1437.8, 1421.0, 1265.5. Anal. Calcd for
Here we provide an effective synthesis which is expected to be
of great value for improved synthesis of new bisindole derivatives
as well as for new development in the field of Fischer indole chem-
istry. Several 1,4-bis(indolin-1-ylmethyl)benzene-based com-
pounds have been assayed for the inhibition of the
physiologically relevant human CA isozymes hCA I and II. These
compounds showed inhibition constants in the range of 39.3–
42.6 lM and 0.17–0.29 lM, against hCA I and hCA II, respectively.
Molecular docking studies of derivatives 9a–c within hCA I and
C32H32N2 (444.26): C, 86.44; H, 7.25; N, 6.30. Found: C, 86.43; H,
7.24; N, 6.29.
hCA II were also carried out and supported the kinetic assays.
4.2.5. 1,4-bis((7,8,9,10-Tetrahydrocyclohepta[b]indol-5(6H)-
yl)methyl)benzene (9c)
4. Experimental
4.1. Chemicals
Light green crystals (2.7 g, 91%) from EtOAc/Hexane; mp 179–
180 °C; 1H NMR (400 MHz, CDCl3) d (ppm): d 7.56–7.51, (m, ArH,
2H), 7.21–7.07 (m, ArH, 8H), 6.89 (s, ArH, 4H), 5.27 (s, CH2, 4H),
2.91–2.85 (m, CH2, 4H), 2.78–2.72 (m, CH2, 4H), 1.89–1.70 (m,
CH2, 12H). 13C NMR (50 MHz, CDCl3) d (ppm): 140.74, 139.49,
137.78, 130.08, 128.31, 122.55, 120.93, 119.64, 116.30, 110.98,
47.91, 33.57, 30.38, 29.01, 28.46, 26.39. IR (KBr, cmꢀ1): 3019.6,
2919.6, 2851.5, 1770.62, 1683.9, 1605.6, 1467.6, 1443.4, 1415.4,
1345.5, 1317.5. Anal. Calcd for C34H36N2 (472.29): C, 86.40; H,
7.68; N, 5.93. Found: C, 86.39; H, 7.67; N, 5.92.
Sepharose 4B, protein assay reagents, 4-nitrophenylacetate and
chemicals for electrophoresis were purchased from Sigma–Aldrich
Co. All other chemicals were of analytical grade and obtained from
Merck.
4.2. Synthesis
4.2.1. Synthesis of 1,4((1-phenylhydrazine)methyl)benzene
A solution of 5 (1 g, 9.24 mmol) in dry THF cooled ꢀ20 °C after
added NaH (225 mg, 9.35 mmol) mixture was stirred at ꢀ20 °C for
2 h under N2. Then (1.2 g, 4.56 mmol) 1,4-bis(bromomethyl)ben-
zene was added and mixture was stirred at room temperature
for 12 h. After removal of the solvent, the residue was dissolved
EtOAc (50 mL) and washed with water (2 ꢁ 50 mL), dried over
MgSO4, and solvent was evaporated. The product 7 was obtained
quantitative (yield yellow oil,1.3 g, 90%).
4.3. Purification of carbonic anhydrase isoenzymes from human
and bovine by affinity chromatography
Purification of hCA I and hCA II were previously described.13–15
4.4. CA inhibition
Carbonic anhydrase activity was assayed by following the
change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to
4-nitrophenylate ion over a period of 3 min at 25 °C using a spec-
1H NMR (400 MHz, CDCl3) d (ppm): d 7.32–7.22 (m, ArH, 8H),
7.09 (d, J = 0.4 Hz, ArH, 4H), 6.83 (t, J = 0.4 Hz, ArH, 2H), 4.61 (s,
4H, CH2), 3.70 (s, 4H, NH2). 13C NMR (50 MHz, CDCl3)
d
trophotometer according to the method described by Verpoorte
21a
(ppm):153.82, 138.88, 131.09, 130.26, 120.65, 115.67, 62.12. IR
(KBr, cmꢀ1): 3327.7, 3182.1, 3020.6, 2918.8, 2829.1, 1595.4,
1495.8, 1350.0, 1220.5, 1152,4, 989.1. Anal. Calcd for C20H22N4
(318.18): C, 55.68; H, 6.96; N, 17.60. Found: C, 55.69; H, 6.95; N,
17.58.
et al.
The enzymatic reaction, in a total volume of 3.0 mL, con-
tained 1.4 mL 0.05 M Tris–SO4 buffer (pH 7.4), 1 mL 3 mM 4-
nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution. A ref-
erence measurement was obtained by preparing the same cuvette
without enzyme solution. The inhibitory effects of compounds 9a–
c is compared with indole. All compounds were tested in triplicate
at each concentration used. Different inhibitor concentrations were
used. Different inhibitor concentrations were used. Control cuvette
activity in the absence of inhibitor was taken as 100%. For each
inhibitor an Activity %–[Inhibitor] graph was drawn. The curve-fit-
ting algorithm allowed us to obtain the IC50 values, working at the
lowest concentration of substrate of 0.15 mM, from which KI val-
4.2.2. General procedure for bisindalizations
A
solution of ketone (6.28 mmol), 1,4((1-phenylhydr-
azine)methyl)benzene (3.14 mmol) and TFA (five drops) in acetic
acid (20 mL) was refluxed for 2 h and cooled to room temperature.
The reaction mixture was evaporated to remove glacial acetic acid
under reduced pressure, crude product was dissolved EtOAc
(100 mL) then washed with water (1 ꢁ 50 mL) and (2 ꢁ 50 mL),
dried over MgSO4 and solvent evaporated The residues were
recrystallized from the below giving solvent mixture for every
molecules, yield (89–92%).
21b
ues were calculated by using the Chenge–Prusoff equation.
The catalytic activity (in the absence of inhibitors) of these en-
zymes was calculated from Lineweaver–Burk plots, as reported
earlier, and represent the mean from at least three different deter-
minations. The enzymes used here were purified from human
21c
4.2.3. 1,4-bis((2,3-Dihydrocyclopenta[b]indol-4(1H)-
blood as described earlier.
yl)methyl)benzene (9a)
Light green crystals (2.4 g, 92%) from EtOAc/Hexane; mp 147–
148 °C; 1H NMR (400 MHz, CDCl3) d (ppm): d 7.49–7.44 (m, ArH,
2H), 7.21–7.15 (m, ArH, 2H), 7.12–7.06 (m, ArH, 4H), 7.02 (s, ArH,
4H), 5.19 (s, CH2, 4H), 2.92–2.86 (m, CH2, 4H), 2.80–2.73 (m, CH2,
4H), 2.60–2.49 (m, CH2, 4H). 13C NMR (50 MHz, CDCl3) d (ppm):
148.09, 143.19, 139.28, 129.00, 126.89, 122.16, 121.19, 120.59,
120.41, 111.77, 49.89, 30.38, 27.12, 26.55. IR (KBr, cmꢀ1): 3025.2,
2924.4, 2845.9, 1675.5, 1611.2, 1580.4, 1457.7, 1435.0, 1376.2,
1345.5, 1163.6, 1018.8. Anal. Calcd for C30H28N2 (416.23): 86.50;
H, 6.78; N, 6.72. Found: C, 86.49; H, 6.79; N, 6.73.
4.5. Protein determination
Protein during the purification steps was determined spectro-
photometrically at 595 nm according to the Bradford method,
using bovine serum albumin as the standard.21d
4.6. SDS polyacrylamide gel electrophoresis
SDS polyacrylamide gel electrophoresis was performed after
purification of the enzymes. It was carried out in 10% and 3%

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taining 0.1% SDS according to Laemmli method.21e
4.6.1. In silico docking studies
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18b
L-His, PDB code 2FW4)
or inhibitors (the hCA II–phenol adduct
PDB code 2HNC)13 were used for the molecular docking calculations
of compounds 9a–c within the active sites of the hCA I and II. Expli-
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calculations. Before the docking simulations, the target structure
were submitted to the protein preparation module of the Schro-
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geometry optimization was performed for these ligands using Po-
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Chem. 2010, 18, 3559; (b) Senturk, M.; Ekinci, D.; Alici, H. A.; Beydemir, S.
Pesticide Pestic. Biochem. Physiol. 2011, 101, 206.
lak-Ribiere
conjugate
gradient
(PRCG)
minimization
(0.0001 kJ Åꢀ1 molꢀ1, convergence criteria).17 Protonation states of
ligands and residues were tested using LigPrep and Protein Prepara-
tion modules under Schrodinger package19–22 at neutral pH (exper-
imentally the compounds have been tested at pH of 7.4). Genetic
Optimisation for Ligand Docking (GOLD) program is used.22 In dock-
ing the maximum number of generic algorithm runs was set to 100
for each compound. The default generic algorithm parameters were
selected (100 population size, 5 number of islands, 100,000 number
of generic operations and 2 for the niche size). Default cutoff values
of 2.5 Å (dH-X) for hydrogen bonds and 4.0 Å for van der Waals dis-
tance were employed. The two scoring functions (GoldScore Fitness
and the ChemScore) were used.22 The GoldScore Fitness function is a
molecular mechanics-like function with four terms:
GoldScore Fitness ¼ SHB-ext þ SVDW-ext þ SHB-int þ SVDW-int
where SHB-ext is the protein–ligand hydrogen-bond score and SVDW-
ext is the protein–ligand van der Waals score. SHB-int is the contribu-
tion to the Fitness due to intramolecular hydrogen bonds in the li-
gand; SVDW-int is the contribution due to intramolecular strain in the
ligand.
On the other hand, the ChemScore function estimates the free
energy of binding of the ligand to a protein as follows:
D
Gbinding
¼
DG0 þ
D
GHBondSHBond
þ
D
GmetalSmetal
þ
DGlipoSlipo
þ
DGrotHrot
where SHbond, Smetal, and Slipo are scores for hydrogen-bonding,
acceptor-metal, and lipophilic interactions, respectively. Hrot is a
score representing the loss of conformational entropy of the ligand
upon binding to the protein. The final ChemScore value is obtained
by adding in a clash penalty and internal torsion terms, which mil-
itate against close contacts in docking and poor internal conforma-
tions. Covalent and constraint scores may also be included:
ChemScore ¼ Gbinding þ Eclash þ Eint
D
Acknowledgments
The authors are grateful to Karamanoglu Mehmetbey University
Scientific Research Council, (BAP) (Project No.: BAP-24-M-11) for
(O.T.).
16. (a) Temperini, C.; Innocenti, A.; Scozzafava, A.; Parkkila, S.; Supuran, C. T. J. Med.
Chem. 2010, 53, 850; (b) Maresca, A.; Temperini, C.; Vu, H.; Pham, N. B.;
Poulsen, S. A.; Scozzafava, A.; Quinn, R. J.; Supuran, C. T. J. Am. Chem. Soc. 2009,
131, 3057; (c) Maresca, A.; Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava,
A.; Supuran, C. T. J. Med. Chem. 2010, 53, 335.
Supplementary data
17. Schrodinger Suite, L.L.C. Schrodinger, New York, USA 2007, (web page:
www.schrodinger.com).
18. (a) Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R.; Farid, R. J. Med. Chem.
2006, 49, 534; (b) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.;
Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. J. Med. Chem.
2006, 49, 6177.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.027.
19. Temperini, C.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2006, 16,
5152.
References and notes
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