Synthesis, characterization, and SAR of arylated indenoquinoline-based cholinesterase and carbonic anhydrase inhibitors

Article abstract of DOI:10.1002/ardp.201800167

We report the synthesis of bromoindenoquinolines (15a–f) by Friedlander reactions in low yields (13–50%) and the conversion of the corresponding phenyl-substituted indenoquinoline derivatives 16–21 in high yields (80–96%) by Suzuki coupling reactions. To explore the structure–activity relationship (SAR), their inhibition potentials to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and human carbonic anhydrase cyctosolic (hCA I and II) enzymes were determined. Monophenyl (16–18) indenoquinolines significantly inhibited the AChE and BChE enzymes in ranges of IC₅₀ 37–57 nM and 84–93 nM, respectively, compared with their starting materials 15a–c and reference compounds (galanthamine and tacrine). On the other hand, these novel arylated indenoquinoline-based derivatives were effective inhibitors of the BChE, hCA I and II, BChE and AChE enzymes with K_i values in the range of 37 ± 2.04 to 88640 ± 1990 nM for AChE, 120.94 ± 37.06 to 1150.95 ± 304.48 nM for hCA I, 267.58 ± 98.05 to 1568.16 ± 438.67 nM for hCA II, and 84 ± 3.86 to 144120 ± 2910 nM for BChE. As a result, monophenyl indenoquinolines 16–18 may have promising anti-Alzheimer drug potential and 3,8-dibromoindenoquinoline amine (15f) can be novel hCA I and hCA II enzyme inhibitors.

Full text of DOI:10.1002/ardp.201800167

Received: 1 June 2018
Revised: 7 July 2018
Accepted: 11 July 2018
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DOI: 10.1002/ardp.201800167
FULL PAPER
Synthesis, characterization, and SAR of arylated
indenoquinoline-based cholinesterase and carbonic
anhydrase inhibitors
Makbule Ekiz1
Ahmet Tutar1
Salih Ökten2
Burcu Bütün3
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Ümit M. Koçyiğit4
Parham Taslimi5
Gülaçtı Topçu6
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1 Faculty of Art and Science, Department of
Chemistry, Sakarya University, Serdivan,
Turkey
We report the synthesis of bromoindenoquinolines (15a–f) by Friedlander reactions
in low yields (13–50%) and the conversion of the corresponding phenyl-substituted
indenoquinoline derivatives 16–21 in high yields (80–96%) by Suzuki coupling
reactions. To explore the structure–activity relationship (SAR), their inhibition
potentials to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and
human carbonic anhydrase cyctosolic (hCA I and II) enzymes were determined.
Monophenyl (16–18) indenoquinolines significantly inhibited the AChE and BChE
enzymes in ranges of IC50 37–57 nM and 84–93 nM, respectively, compared with
their starting materials 15a–c and reference compounds (galanthamine and tacrine).
On the other hand, these novel arylated indenoquinoline-based derivatives were
effective inhibitors of the BChE, hCA I and II, BChE and AChE enzymes with Ki
values in the range of 37 2.04 to 88640 1990 nM for AChE, 120.94 37.06 to
1150.95 304.48 nM for hCA I, 267.58 98.05 to 1568.16 438.67 nM for hCA II,
2 Faculty of Education, Department of Maths
and Science Education, Kırıkkale University,
Kirikkale, Turkey
3 Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Bezmialem Vakif
University, Istanbul, Turkey
4 Vocational School of Health Services,
Cumhuriyet University, Sivas, Turkey
5 Faculty of Science, Department of
Chemistry, Ataturk University, Erzurum,
Turkey
6 Faculty of Pharmacy, Department of
Pharmacognosy/Phytochemistry, Bezmialem
Vakif University, Istanbul, Turkey
Correspondence
and 84 3.86 to 144120 2910 nM for BChE. As
a result, monophenyl
Dr. Salih Ökten, Division of Science
Education, Faculty of Education, Department
of Maths and Science Education, Kirikkale
University, Yahsihan, Kirikkale 71450, Turkey.
Email: salihokten@kku.edu.tr
indenoquinolines 16–18 may have promising anti-Alzheimer drug potential and
3,8-dibromoindenoquinoline amine (15f) can be novel hCA I and hCA II enzyme
inhibitors.
Prof. Ahmet Tutar, Faculty of Art and Science,
Department of Chemistry, Sakarya University,
Serdivan, Sakarya 54187, Turkey.
K E Y W O R D S
acetylcholinesterase, bromoindenoquinolines, butyrylcholinesterase, carbonic anhydrase,
enzyme inhibition, phenyl indenoquinolines, SAR
Email: atutar@sakarya.edu.tr
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1
INTRODUCTION
plaques, the major pathological sign in the brain of AD patients, is a
degradation product of cholinesterase (ChE) enzyme.[2] Currently,
Alzheimer's disease (AD) is acknowledged as progressive multifarious
neurodegenerative disorder[1] and considered as lack of cognitive
abilities, i.e., thinking and judging, in the elder people.[1,2] AD is
accompanied by an increase of acetylcholinesterase (AChE) activity
that leads to run out of cholinergic neurotransmission in the brain areas
related to memory and learning.[3] Amyloid beta-protein (A beta)
there are several approved drugs as AChE inhibitors (AChEIs) including
donepezil, rivastigmine, and galanthamine for the treatment of AD.[4,5]
These drugs increase acetylcholine (ACh) level in the central nervous
system to enhance cholinergic neurotransmission.[6] The inhibition of
both hydrolysis products of ACh, AChE and butyrylcholinesterase
(BChE), is essential for successful treatment of AD because recent
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Arch Pharm Chem Life Sci. 2018;e1800167.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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studies in AD patients reported that while the AChE activity is
significantly reduced in specific regions of the brain, BChE activity can
increase.[3,7,8] Most preferred medication is discovery of either new
cholinesterase inhibitors or N-methyl-D-aspartate receptor antago-
nists such as memantine.[9] Adverse effects of some synthetic AChEIs
used for the treatment of AD such as tacrine and donepezil let us to
find innocent and most potent derivatives of those drugs.[10]
Therefore, it is very important to find new substances, more efficient
and less expensive than the currently used drugs is urgently needed.
Carbonic anhydrase (CA, EC 4.2.1.1) isoforms catalyze a simple,
yet decisive, reaction: the reversible hydration of water and carbon
dioxide to proton and a bicarbonate anion.[11–13] This reaction, in the
absence of CA cannot proceed with a perceptible rate under
physiological positions. CAs are ubiquitous metalloenzymes compris-
ing Cd(II)-, Zn(II)-, or Fe(II)-, available in eukaryotes and prokaryotes,
and classified at least seven class (α-CA, β-CA, δ-CA, γ-CA, ζ-CA, η-CA,
θ-CA).[14] CA isoforms play a momentous role in multitude physiologi-
cal activities of eukaryotes such as CO2 transport, respiration,
photosynthesis between others and electrolyte secretion.[15] Addi-
tionally, to the defined role of CA inhibitors (CAIs) as antiglaucoma
drugs and diuretics, it has newly appointed that they have potential as
antiobesity, anticonvulsant, anti-infective drugs, and anticancer.[16]
The quinoline skeleton occurs in several natural compounds and
pharmacologically active substances displaying a broad range of
biological activity.[17–20] In addition to the medicinal uses, quinolines
have been employed in the study of bioorganic and bioorganometallic
processes. The Friedlander reaction is a well-known method for
preparing quinolines,[21] it is still considered as one of the most useful
methods for preparing quinolines and related bicyclic azaaromatic
compounds. Indenoquinolines are quinoline derivatives bearing tetra-
aromatic heterocycle and distinguished as profound chemical and
biological agents due to diverse range of their biological activities such
as 5-HT-receptor binding and anti-inflammatory activities.[22] They
have also acted as antitumor agents,[23,24] steroid reductase inhib-
itors,[25] new potential topo I/II inhibitors,[26] antimalarials,[27] and also
AChEIs.[2,28] Thus, the synthesis of these molecules has attracted
considerable attention. However, there are restricted reports about
synthesis of substituted indenoquinoline amine, displayed AChE
inhibitory activity. Some reported studies showed that halogenated
indenoquinoline analogues have better inhibitory activity compared
with unsubstituted ones.[28] This study reported that especially fluoro
substituted at C-2 position of benzene ring fused 1-H indene having
more AChE inhibitory activity potential compared with methoxy,
chloro-substituted indenoquinoline amine derivatives.[28]
Recently, we have reported the preparation of novel tricyclic
tacrine derivatives, including five-, six-, and seven-membered hydro-
cycles,[31] through cyclodehydration reaction of several ketones and
2-amino-3,5-dibromobenzonitrile via Friedlander reaction furnished a
series of dibrominated tacrines. Then bromotacrine analogues were
converted to corresponding disubstituted tacrine analogues by metal
catalyzed or metal halogen exchange. In continuation of our research
on bioactive molecules, we designed and synthesized a new series of
bromo- and phenyl-substituted indenoquinoline amine. Due to having
the AChE, hCA I and hCA II inhibition potentials of the tricyclic
structure of the 1,2,3,4-tetrahydroacridine nucleus,[37,38] we ex-
panded to tetracyclic ones. Thus, this work was to explore
the synthesis of novel bromoindenoquinoline amines via
Friedlander reaction between 2-amino-3,5-dibromobenzonitrile (7)
and 2-amino-5-bromobenzonitrile (11) and bromoindan-1-ones (12
and 13) in the presence of some Lewis acids. In this series, we
synthesized phenyl-substituted indenoquinoline amine analogues by
Suzuki coupling reactions. In addition, we focused on investigation of
anti-Alzheimer activities of novel-synthesized tetracyclic polyfunc-
tional indenoquinoline amine as potentials of AChE and BChE
inhibitors. Although there is no association between carbonic
anhydrase inhibition and Alzheimer, as another study, we determined
their carbonic anhydrase isoforms, the cytosolic hCA I and II inhibition
activities. Furthermore, to fill the gap in the structure–activity
relationship (SAR) of substituted indenoquinoline amine derivatives,
IC50 values for the inhibition of AChE, BChE, hCA I and hCA II were
studied by means of SAR approach, in search for describing the
dependence of AChE and BChE inhibitory activities on substituents of
indenoquinoline amine cycles.
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
In our previous papers,[31,36] one-pot synthesis was described for
tricyclic bromotacrine derivatives (1–6) (Figure 1) using Friedlander
reactions of several cyclic ketones and brominated 2-amino-3,5-
dibromobenzonitrile (12) in the presence of some Lewis acids. Then,
tacrine bromides (2 and 3) were converted corresponding disubsti-
tuted tacrine derivatives via metal-halogen and copper-assisted
substitution reactions.[31] Furthermore, the evaluation of their anti-
Alzheimer and anticarbonic anhydrase potentials indicated that
methoxy (10), cyano (7), silyl (6 and 9), and thiomethyl (8) tacrine
Halogenated aromatic compounds, including quinoline,[29,30]
tacrine,[31] anthracene,[32] indane,[33] and naphthalenes,[34,35] would
enable the synthesis of diverse aromatic frameworks because the
halogen, especially bromine, could enhance biological activity in many
cases and could also be used for further functionalization in preparing
other molecules.[31] For quinoline-based compounds, tacrine, inden-
oquinoline, etc., ring substitution by halogen is rather complex.[31,36]
Particularly, direct bromination of polycyclic aromatics with
N-function is difficult due to formation of N-bromine complex.[29]
FIGURE 1 Tricyclic disubstituted tacrine derivatives (1–10)

EKIZ ET AL.
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derivatives had selective AChE and BChE inhibitory activities
and showed significant inhibition against hCA
enzymes.[37,38]
I and hCA II
In view of these observations and in continuation of our
current interest in the synthesis of disubstituted tacrines for
biological evaluations,[38,39] the promising AChE and BChE
inhibitory features of disubstituted tacrine derivatives have
encouraged us to design and synthesize tetracyclic indenoquino-
line amine bearing phenyl ring 16–21 for anti-Alzheimer properties
as depicted in Figure 2. We have planned to investigate anti-
Alzheimer and carbonic anhydrase activities of the novel-
synthesized compounds 16–21 against AChE and BChE and the
cytosolic hCA I and hCA II enzymes.
SCHEME 1 Bromination of 2-aminobenzonitrile 11 and synthesis
of bromoaminobenzonitrile derivatives (12 and 13). Reagents and
conditions; (i) Br2 (2.2 equiv), AcOH, 10–15°C, 10 min; (ii) Br2 (1.1
equiv), AcOH, 10–15°C, 10 min
mass spectrometry. The 1H NMR spectra of compounds 15a–i, which
consisted of aryl Hs, alkyl Hs (δ 3.79–3.99 ppm), and NH signals
matched quite well with the suggested structures. For example, the
1H NMR spectra of 15g–i clearly show tribromo structures due to two
aryl signals with meta coupling constants (4J = 1.8 Hz). One aliphatic
[3.84 (s), 3.99 (s), and 3.88 (s), respectively] and NH2 shifts [8.35–8.68
(brs)] are recorded for each 15g–i. In the 13C NMR spectra of
compounds 15g–i, one carbon resonates in the sp3 region and 15
carbon resonances in the sp2 region are also in agreement with the
proposed structures.
To achieve this goal, functional bromo tetracyclic indenoquinoline
amine derivatives were obtained because heterocyclic aromatics are
key structures in
a large amount of pharmacological com-
pounds.[19,20,40] Due to that the direct bromination leads to some
problems in obtaining brominated N function aromatics.[29,31,36] We
attempted Friedlander reactions between 2-aminobenzonitrile (11) or
corresponding brominated 2-aminobenzonitriles (12 and 13) and
bromoindan-1-ones (14a–c), instead of direct bromination of inden-
oquinoline nucleus. First, the reaction of 2-aminobenzonitrile (11) with
Br2 was examined in AcOH at 10–15°C. After the reaction between
2-aminobenzonitrile and 1 equivalent of Br2, the 1H NMR spectra of
the mixture of products indicated 2-amino-3,5-dibromobenzonitrile
(12) and 2-amino-5-bromobenzonitrile (13). Then the products were
efficiently isolated with column chromatography. Compound 12 was
furnished with treatment of 2 equivalents of Br2 in 98% yield as the
sole product (Scheme 1).
To demonstrate the value of brominated indenoquinoline amines
15a–f as starting materials for numerous useful compounds, we
investigated their Suzuki Miyaura cross-coupling reactions. These
coupling reactions of monobromo 15a–c and dibromoindenoquinoline
amines 15d–f with different equivalents of phenylboronic acid (1.1 and
2.2 equivalents) afforded 1-phenyl 16, 2-phenyl 17, 3-phenyl 18,
1,8-diphenyl 19, 2,8-diphenyl 20, and 3,8-diphenyl 21 indenoquinoline
amines in high yields (80–96%) (Scheme 3 and Table 1). The reactions
were carried out with Pd(PPh3)4 (5 mol%) as the catalyst and aq
Na2CO3 (3 M) as the base. 1,4-Dioxane was used as the solvent and the
reactions were performed at 100°C for 4 h.
The cyclodehydration reaction under toluene reflux was
further evaluated with several bromoindan-1-ones 14a–c and
2-aminobenzonitrile (11) in the presence of InCl2, Lewis acid as
catalyst. Anthranilonitrile (2-aminobenzonitrile, 11) and 4-bromo 14a,
5-bromo 14b, and 6-bromoindan-1-one (14c), instead of monocyclic
ketones in our previous study,[31] furnished moderate yields of
products 15a (50%), 15b (48%), and 15c (44%), respectively (Scheme 2).
Furthermore, 2-amino-5-bromobenzonitrile (13) and 2-amino-3,5-
dibromobenzonitrile (12) with corresponding bromoindan-1-ones
14a–c under the same reaction conditions furnished dibromo
(15d–f) and tribromo (15g–i) indenoquinoline amines with low yields
(35, 30, 23, 20, 13, and 15%, respectively) (Scheme 2).
Structural characterization of the phenyl-substituted indenoqui-
noline amines 16–21 was further confirmed by elemental analysis and
1H NMR, 13C NMR, and FT-IR spectra. In the 1H NMR of phenyl
derivatives 16–21 provided new aromatic signals at δH 7.33–7.73 (m),
7.40–7.72 (m), 7.37–7.82 (m), 7.43–7.90 (m), 7.38–7.79 (m), 7.32–7.76
(m), respectively. In addition, 13C NMR spectra of 16–21, consisting of
new sp2 resonances supported the proposed structures.
Structures of bromoindenoquinoline amines (15a–i) were deter-
mined by 1H NMR, 13C NMR, FT-IR spectroscopy, and high resolution
SCHEME 2 Synthesis of mono- (15a–c), di- (15d,e), and tribromo
(15g,h) indenoquinolines. Reagents and conditions; (i) toluene, InCl3
(1.1 equiv), reflux, 24 h, then NaOH (2 M), reflux, 24 h
FIGURE 2 Design strategy for new indenoquinoline amines

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EKIZ ET AL.
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SCHEME 3 Synthesis of phenyl-substituted (16–21) indenoquinoline amines. Reagents and conditions; (i) PhB(OH)2 (1.3 equiv), Pd(PPh3)4
(0.05 equiv), K2CO3 (3 M), dioxane, reflux, 4 h. (ii) PhB(OH)2 (2.6 equiv), Pd(PPh3)4 (0.05 equiv), K2CO3 (3 M), dioxane, reflux, 4 h
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2.2 Biological activity
of references compounds, tacrine (IC50 = 59 nM for AChE and
IC50 = 23 nM for BChE, taken in our study) and galanthamine
(IC50 = 1280 nM for AChE and IC50 = 19530 nM for BChE, obtained
in our study) is reported in Table 1, expressed as IC50 values.
The monophenyl-substituted indenoquinoline amines 16–18
were found to be potent inhibitors of cholinesterases with IC50 in
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2.2.1 Anticholinesterase activity results
The inhibitory activity against AChE and BChE of novel-synthesized
bromo- and phenyl-substituted indenoquinoline amine derivatives and
TABLE 1 Structural data and AChE and BChE inhibitory activities (IC50) of novel-substituted indenoquinoline amine derivatives
No.
15a
15b
15c
15d
15e
15f
15g
15h
15i
16
R1
H
R2
H
H
H
H
H
H
Br
Br
Br
H
H
H
H
H
X
Y
Z
Yield (%)
50
Mp (°C)
IC50 for AChE (nM)
2610 170
5700 127
2170 273
230 15
r2
IC50 for BChE (nM)
1340 760
3890 183
46550 2670
2620 390
59790 2140
3660 259
4790 239
38600 2180
2420 283
93 2.39
r2
H
H
Br
H
H
Br
H
H
Br
H
H
Ph
H
H
H
Br
H
227–228
238–239
240–241
291–292
283–284
280–281
297–298
294–295
291–292
234–235
235–236
234–235
251–252
253–254
0.992
0.999
0.998
0.999
0.989
0.992
0.999
0.999
0.998
0.999
0.987
0.998
0.988
0.999
0.999
0.997
0.994
0.999
0.999
0.999
0.999
0.994
0.994
0.999
0.997
0.999
0.998
0.998
0.998
0.989
0.999
0.999
H
Br
H
48
H
H
44
Br
Br
Br
Br
Br
Br
H
H
Br
H
35
Br
H
30
3730 204
23420 1620
5410 1450
88640 1990
3530 117
37 2.04
H
23
H
Br
H
20
Br
H
13
H
15
H
Ph
H
96
17
H
Ph
H
95
57 2.39
87 3.78
18
H
H
92
56 2.46
84 3.86
19
Ph
Ph
H
Ph
H
83
4870 102
1090 175
59 1.47
144120 2910
13780 253
23 1.06
20
Ph
91
Tacrine (reference compound A)
Galantamine (reference compound B)
1280 106
19530 1740

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nanomolar concentration scale. The most active AChE inhibitor in the
assayed series of substituted indenoquinoline amine analogues are
dibromoindenoquinoline amine 15d and monophenyl derivatives
16–18 with IC50 ranging between 37 and 230 nM. The 1,8-dibromo
derivative 15d significantly inhibited AChE enzyme at IC50 of 230 nM,
while 3,8-dibromo 15f (IC50 = 23420 nM) and 2,6,8-tribromoindeno-
quinolines 15h (IC50 = 88640 nM) showed low inhibition against AChE
enzyme compared with galanthamine (Table 1). Despite the high
inhibition of 15d against AChE enzyme, this compound moderately
inhibited BChE enzyme (IC50 = 2620 nM). According to reported
results, compound 15d proved to be the most selective AChE inhibitor,
compared with galanthamine and unsubstituted tacrine. Furthermore,
the high AChE inhibition potentials of 16–18, as close to IC50 value of
tacrine, indicated that they could be novel promising AChE inhibitors
(Table 1).
ranging 120.94–1150.95 and 267.58–1568.16 nM, respectively.
Acetazolamide (AZA), a clinically used sulfonamide CAI was used in
the assays as a reference drug. As shown in Table 2, 13 compounds
(15a–h and 16–20) were found to have IC50 in the range of 138.63–
1064.51 nM against hCA I. Also, these compounds have IC50 in the
range of 233.41–1300.18 nM against hCA II. All these compounds
displayed good activity against hCA I and hCA II as compared to AZA
with IC50 values of 3745.94 and 3347.82, respectively (Table 2).
Among all the above compounds, only compound 15f displayed
significant activity, i.e., IC50 138.63 and 213.66 nM, as compared to
AZA. Moreover, compounds 15a, 15c–e, and 18–20 were found to
possess good activity against hCA I, i.e., IC50 330.76 nM in agreement
with AZA. Although all other remaining compounds were found to
possess moderate activity, their inhibition activities more than as AZA
against hCA I. Furthermore, all tested compounds showed inhibition
activity against hCA II in the same manner as hCA I, but in higher
concentrations, i.e., IC50 543.18 nM (Table 2). This study clearly
indicates that bromo- and phenyl-substituted indenoquinoline amine
derivatives (15a–h, 16–20) could be promising hCA I and hCA II
inhibitor candidates.
The BChE inhibition results showed that the most active
compounds are also monophenyl indenoquinoline amines 16–18
with IC50 values of 93, 87 and 84 nM, respectively. Furthermore, most
tested compounds showed good inhibition against BChE with IC50
ranging between 1340–13780 nM even better than standard
compound galanthamine (IC50 = 19530 μM), except for compounds
15c (IC50 = 46550 nM), 15e (IC50 = 59790 nM), 15h (IC50 = 38600 nM)
and 19 (IC50 = 144120 nM).
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2.2.3 The SAR study for enzyme inhibition
Substituents were introduced in synthetically accessible positions of
the aryl rings indenoquinoline amine as well as on primary amino
group in order to investigate the SAR of the series. The structural
characteristics of novel-substituted indenoquinoline amines and
AChE and BChE inhibition data expressed as IC50 values are shown
in Tables 1 and 2. Also hCA I and II inhibitory potentials’ data
expressed as IC50 and Ki values in nM concentrations are shown in
Table 2.
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2.2.2 Anticarbonic anhydrases activity results
The substituted indenoquinoline amine derivatives reported here were
determined as inhibitors of two physiologically relevant CA isoforms,
the cytosolic hCA I and II by an esterase assay.[41] The hCA I and II
isoenzymes were inhibited by the novel-synthesized bromo- and
phenyl-substituted indenoquinoline amine derivatives, with Ki values
TABLE 2 The hCA I and hCA II inhibitory activities (IC50 and Ki) of novel-substituted indenoquinoline amine derivatives
IC50 (nM)
Ki (nM)
Compound no.
hCA I
r2
hCA II
r2
hCA I
hCA II
15a
15b
15c
15d
15e
15f
15g
15h
16
330.76
693.06
409.08
403.61
428.33
138.63
863.11
1213.66
1051.58
1064.51
402.22
421.02
415.77
3745.94
0.9869
0.9778
0.9861
0.9595
0.9661
0.9885
0.9884
0.9854
0.9800
0.9709
0.9834
0.9773
0.9771
0.9653
612.18
734.58
620.41
548.66
560.67
233.41
1300.18
579.43
797.46
889.62
502.92
535.96
427.75
3347.82
0.9436
0.9579
0.9572
0.9735
0.9858
0.9621
0.9149
0.9238
0.9368
0.9139
0.9574
0.9663
0.9642
0.9899
322.75 52.58
543.18 91.98
573.33 196.12
377.08 72.21
332.97 47.37
120.94 37.06
587.03 100.71
1150.95 304.48
986.01 254.22
1131.54 277.61
341.88 42.02
526.27 224.21
408.06 124.27
3320.46 716.02
682.22 72.85
580.98 132.22
495.68 136.84
495.02 94.08
714.07 315.33
267.58 98.05
1568.16 438.67
720.88 210.97
762.88 270.16
1447.70 522.32
677.93 244.07
421.63 87.32
491.18 171.03
2718.92 531.18
17
18
19
20
AZA

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It is evident that substituents at C-1, C-2, C-3, and C-8 for
indenoquinoline amines present various inhibitory activity. When
comparing the compounds containing the Br substituent with different
positions and the numbers, it is observed that Br at both C-1 and C-8
(IC50 230 nM) increased the inhibition of AChE activity for dibromo
compounds (15d–f); however, this substituent at both C-3 and C-8
(IC50 23420 nM) led to reduction in the activity. The only one bromine
atom of monobromides 15a–c at C-1, C-2, or C-3 positions did not
increase the AChE and BChE inhibition activities, in particular, Br at C-3
in the indane backbone significantly reduced the BChE inhibition
activity (IC50 46550 nM). Furthermore, the presence of Br substituent
at the positions of C-6, C-8, and anywhere in the benzene ring fused
penta atomic cycle (C-1, C-2, or C-3) in tribromoindenoquinolines
(15g–i) decreased the both AChE and BChE inhibition activities, i.e.,
IC50 values of 15h 88640 nM for AChE and 38600 nM for BChE. The
other interesting observation is that Br atom in the presence at the
position of C-1, C-2, or C-3 for dibromoindenoquinoline is crucially
important for cholinesterase inhibition activity. The bromine at C-1 in
15d significantly inhibited AChE (IC50 230 nM) and moderately
inhibited BChE (IC50 2620 nM), while Br at C-3 in 15f and Br at C-2
in 15e significantly reduced inhibitions of AChE (IC50 23420 nM) and
BChE (IC50 59790 nM), respectively.
BChE inhibitory activities than bromo-substituted ones. The position
and kinds of substituents are important to anti-AChE and BChE
activities.
|
3
CONCLUSION
In this work, we have decribed a convenient route to synthesize
bromoindenoquinoline amines (15a–h) by Friedlander reactions
starting with 2-aminobenzonitrile (11) and its brominated derivatives
12 and 13. In addition, we showed a simple synthetic methodology
to obtain phenyl-substituted indenoquinolines by Suzuki cross-
coupling reactions between bromoindenoquinolines 15a–f and
phenylboronic acid. Several bromo- and phenyl-substituted ana-
logues were found to be potent and selective inhibitors of AChE,
BChE, hCA I and hCA II in nanomolar concentration scale. Moreover,
we attempted to determine the SAR of the inhibition effects of the
introduction of the bromo and phenyl substituents in synthetically
accessible positions of tetracyclic nucleus. In case of the presence of
phenyl groups at only C-1, C-2, or C-3, the inhibition activities of
arylated indenoquinolines against cholinesterase enzymes (AChE
and BChE) were found the highest potency. On the contrary, the
phenyl bound to positions of both C-1 and C-8 significantly reduced
the inhibition. Furthermore, the inhibition effect of two Br atoms
bound at both C-2 and C-8 increased, but three bromine atom at C-
2, C-6, and C-8 led to significant decrease of the inhibition activity.
In conclusion, monophenyl-substituted indenoquinoline ana-
logues have been potential of cholinesterase inhibitors. In addition,
bromoindenoquinoline have potential to convert novel cholinesterase
inhibitor by substitution or coupling reactions. This promising result
can contribute to the investigation of a cure for Alzheimer or therapy in
the future.
The introduction of phenyl substituent on compounds 16–18 led to
incredibly increased inhibition activities against AChE and BChE,
compared with their starting materials (15a–c). Also, these compounds
(16–18) (average IC50 = 50 nM for AChE and 88 nM for BChE) displayed
similar inhibition activities to tacrine. The significant increase on
inhibition activity was observed in case of phenyl groups introduced
at C-1, C-2, or C-3 positions of benzene ring fused penta atomic cycle in
16–18. Actually, the phenyl substituent bounded at C-2 and C-8 in
diphenyl indenoquinoline 20 slightly increased the inhibition activity
against both ChE enzymes (IC50 = 1090 nM for AChE and 13780 nM for
BChE), however, the phenyl groups at C-1 and C-8 in compound 19
significantly decreased the inhibition, especially against BChE
(IC50 = 144120 nM), compared with starting compound 15d
(IC50 = 2620 nM).
|
4
EXPERIMENTAL
|
4.1 Chemistry
When comparing hCA
I and II inhibition activities of the
indenoquinolines containing the Br and phenyl substituent with the
samepositions,itisobservedthatthepresenceofBratbothC-3andC-8
|
4.1.1 General
Thin layer chromatography was carried out on Merck silica F254
0.255 mm plates and spots were visualized by UV at 254 nm.
Flash column chromatography was performed using Merck 60
(70–230 mesh) silica gel. Melting points were determined on a
MPM-H1 capillary melting points apparatus. Solvents were concen-
trated at reduced pressure. IR spectra were recorded on a Shimadzu
Prestige-21 (200 VCE) FT/IR instrument. Mass spectra were recorded
on an Agilent 6530 Accurate-Mass spectrometer under electron-
impact (EI) and chemical ionization conditions. Elemental analysis was
recorded on an Elementar Vario MICRO Cube instrument. NMR
spectra were recorded on a Varian Infinity Plus at 300 MHz and on an
Agilent at 600 MHz for 1H and at 75 MHz for 13C NMR.
indenoquinoline nucleus displayed high inhibition against hCA
I
(IC50 = 138.63 nM) and hCA II (IC50 = 233.41 nM), compared with the
other derivatives and AZA. The evaluation of bromine in tribromides
(15g,h) indicated that the bromine atom at C-1 in 15g reduced the
inhibition activity against hCA II (IC50 = 1300.18 nM) while Br at C-1 in
15h led to show low inhibition against hCA I (IC50 = 1213.66 nM)
enzyme. Furthermore, the phenyl groups did not show significant effect
on inhibition activity. However, the presence of phenyl groups at only
C-1 or C-2 in compounds 16 and 17 causes to have lower inhibition
effect against hCA I (IC50 = 1051.58 and 1064.51 nM, respectively),
compared with monobromoindenoquinoline analogues (15a,b)
(IC50 = 330.76 and 639.06 nM, respectively).
The NMR spectra and the InChI codes of the investigated
compounds are provided as Supporting Information.
These results indicated that phenyl substitutent at position of C-1/
C-2/C-3 for indenoquinoline derivatives are more critical for AChE and

EKIZ ET AL.
7 of 11
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|
4.1.2 Synthesis of 2-amino-3,5-dibromobenzonitrile
(12)
compound (15a) (0.51 g, 50%). Light yellow powder solid, mp 227–
228°C. 1H NMR (300 MHz, DMSO-d6) δ 3.80 (s, 2H, CH2), 6.95 (brs,
2H, NH2), 7.38–7.43 (m, 2H, ArH), 7.60 (t, 3J = 8.4, 3J = 6.9 Hz, 1H,
ArH), 7.65 (d, 3J = 8.1 Hz, 1H, ArH), 7.88 (d, 3J = 8.4 Hz, 1H, ArH), 8.02
(d, 3J = 7.5 Hz, 1H), 8.25 (t, 3J = 8.1, 3J = 8.4 Hz, 1H, ArH). 13C NMR
(75 MHz, DMSO-d6) δ 159.9, 149.7, 147.5, 144.8, 144.1, 132.0, 129.8,
129.3, 129.0, 123.7, 122.8, 120.6, 120.4, 118.4, 112.8, 34.4.
FT-IR (neat v∼ cm−1): 3473–3302 (NH2), 3163 (Arom. C─H),
Bromine (3.52 g, 0.022 mol) was added dropwise to a magnetically
stirred, cooled (ice-water bath) solution of 2-aminobenzonitrile 11
(1.18 g, 10 mmol) in acetic acid (30 mL) at 15–20°C over 10 min. The
reaction mixture was stirred at room temperature for 5 h, then added
to ice-water (100 mL), and the white precipitate was collected by
filtration. The precipitate was washed thoroughly with water and dried
on Na2SO4 at room temperature to afford a white solid mass 12 (98%),
mp = 155–156°C (Lit.,[41] 156°C); Rf (30% EtAcO/hexane) = 0.37.
1H NMR (300 MHz, CDCl3): δ 7.73 (d, 4J = 2.3 Hz, 1H), 7.48 (d,
4J = 2.3 Hz, 1H), 4.90 (brs, 2H) ppm. 13C NMR (75 MHz, CDCl3): δ
146.4, 139.6, 133.8, 115.8, 109.9, 108.4, 98.1 ppm. All data were
identical to those reported in the literature.[40]

2970–2866 (Aliph. C─H), 1641, 1509 (C N), 1439, 1385, 1125,
753–727 (C─Br). HRMS (ESI) m/z: calcd. for
C
16H12N279Br
[M+H]+: 311.0184. Found: 310.9884.
10-Amine-2-bromo-11-H-indeno[1,2-b]quinoline (15b)
Unsubstituted anthranilonitrile (11) (0.24 g, 2.2 mmol), 5-bromoindan-
1-one (14b) (0.46 g, 2.2 mmol), and InCl3 (0.52 g, 2.4 mmol) gave the
title compound (15b) (0.30 g, 48%). Light yellow powder solid, mp
238–239°C. 1H NMR (300 MHz, DMSO-d6): δ 3.96 (s, 2H, CH2), 7.57
(t, 3J = 8.3 Hz, 1H, ArH), 7.66 (d, 3J = 8.7 Hz, 1H, ArH), 7.85 (t, 1H,
3J = 8.5 Hz, ArH), 7.88 (s, 1H, ArH), 7.96 (d, 3J = 8.4 Hz, 1H, ArH), 8.21
(d, 3J = 8.3 Hz, 1H, ArH), 8.45 (d, 3J = 8.8 Hz, 1H, ArH), 8.60 (brs, 1H,
NH), 8.87 (brs, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ 154.2, 151.5,
148.1, 138.8, 134.0, 133.7, 131.6, 129.8, 126.6, 126.1, 125.0, 124.3,
120.4, 116.4, 114.3, 33.9. FT-IR (neat v∼ cm−1): 3330 (NH2), 3066
|
4.1.3 Synthesis of 2-amino-5-bromobenzonitrile (13)
Bromine (1.63 g, 10.2 mmol) was added dropwise to a mechanically
stirred, cooled (ice-water bath) solution of 2-aminobenzonitrile 11
(1.18 g, 10.0 mmol) in acetic acid (15 mL) at 15–20°C in 10 min.
Reaction mixture was stirred at room temperature for 2 h, then added
to ice-water (100 mL) and the white precipitate was collected by
filtration. The precipitate was washed thoroughly with water and dried
on Na2SO4 at room temperature to afford white solid. The TLC analysis
revealed that the final product was a mixture of two products. The
dibromide 12 and monobromide 13 were isolated using a silica gel
column eluting with a mixture of hexane/chloroform (5:1). 12 (0.21 g)
8%, 13 (1.77 g) 90% were isolated in pure forms. White solid powder
13 (90%), mp = 94–95°C; 1H NMR (300 MHz, DMSO-d6): δ 7.58 (d,
J = 2.4 Hz, 1H), 7.41 (dd, J = 9.0, 2.4 Hz, 1H), 6.74 (d, J = 9.0 Hz, 1H),
6.26 (brs, 2H) ppm. 13C NMR (75 MHz, DMSO-d6): δ 151.0, 136.7,
133.9, 117.3, 116.8, 105.2, 95.0 ppm. FT-IR (neat v∼ cm−1): 3434,
3351, 2218, 1629, 1485, 1303, 1255.

(Arom. C─H), 2970 (Aliph. C─H), 1738, 1602, 1508 (C N), 1428,
1384, 965, 822–671 (C─Br). HRMS (ESI) m/z: calcd. for
C
16H12N279Br [M+H]+: 311.0184. Found: 310.9879.
10-Amine-3-bromo-11-H-indeno[1,2-b]quinoline (15c)
Unsubstituted anthranilonitrile (11) (0.35 g, 3.3 mmol), 6-bromoindan-
1-one (14c) (0.7 g, 3.3 mmol), and InCl3 (0.8 g, 3.6 mmol) gave the title
compound (15c) (0.45 g, 44%). Light yellow powder solid, mp 240–
241°C. 1H NMR (300 MHz, DMSO-d6, ppm): δ 3.80 (s, 2H, CH2), 6.88
(brs, 2H, NH2), 7.39 (t, 1H, 3J = 8.4, 3J = 4.0 Hz, ArH), 7.60–7.61 (m, 3H,
ArH), 7.88 (d, 3J = 8.4 Hz, 1H, ArH), 8.09 (s, 1H, ArH), 8.25 (d,
3J = 8.1 Hz, 1H, ArH). 13C NMR (75 MHz, DMSO-d6, ppm): δ 159.2,
149.4, 147.5, 144.3, 144.0, 131.7, 129.3, 129.0, 128.1, 123.9, 123.7,
122.8, 120.7, 118.4, 113.9, 32.4. FT-IR (neat v∼ cm−1): 3298 (NH2),
3075 (Arom. C─H), 2970–2906 (Aliph. C─H), 1738, 1644, 1562
|
4.1.4 General method for the cyclodehydration
reaction
Unsubstituted or brominated anthranilonitrile (1 mmol, 1 equivalent),
bromoindanone (1.0 mmol, 1 equivalent), and sodium-dried toluene
(15 mL) were placed in a three-necked round bottomed flask fitted
with an overhead stirrer. InCl3 as Lewis acid (1.1 mmol, 1.1 equivalent)
was added. The reaction mixture was heated at 120–130°C for 24 h
under stirring. On cooling at rt, the toluene was decanted to liberate
the product and the remaining solids were treated with sodium
hydroxide (2 M, 15 mL) and heated at reflux for 24 h. After cooling, the
organic components were extracted with chloroform, the organic
layers were combined and dried over Na2SO4. The solvent was
evaporated in vacuo to give the desired product.

(C N), 1435, 1363, 883–743 (C─Br). HRMS ESI m/z: calcd. for
16H12N279Br [M+H]+: 311.0184. Found: 310.9890.
C
10-Amine-1,8-dibromo-11-H-indeno[1,2-b]quinoline (15d)
2-Amino-5-bromobenzonitrile (13) (0.35 g, 1.78 mmol), 4-bromoin-
dan-1-one (14a) (0.38 g, 1.78 mmol), and InCl3 (0.51 g, 2.30 mmol)
gave the title compound (15d) (0.24 g, 35%). Light yellow powder
solid, mp 291–292°C. 1H NMR (300 MHz, DMSO-d6, ppm): δ 3.79
(s, 2H, CH2), 7.08 (brs, NH2), 7.42 (t, 3J = 7.5 Hz, 1H, ArH), 7.67–
7.71 (m, 2H, ArH), 7.81 (d, 3J = 9.0 Hz, 1H, ArH), 7.99 (d, 3J = 7.5 Hz,
1H, ArH), 8.52 (d, 4J = 2.0 Hz, 1H, ArH). 13C NMR (75 MHz, DMSO-
d6, ppm): δ 160.7, 148.4, 147.2, 145.1, 143.8, 132.5, 132.3, 131.6,
130.2, 125.4, 121.0, 120.7, 120.0, 116.8, 113.8, 34.7. FT-IR (neat
10-Amine-1-bromo-11-H-indeno[1,2-b]quinoline (15a)
v∼ cm−1
)
3446 (NH2), 3198–3027 (Arom. C─H), 2968–2841
Unsubstituted anthranilonitrile (11) (0.35 g, 3.3 mmol), 4-bromoindan-
1-one (14a) (0.7 g, 3.3 mmol), and InCl3 (0.8 g, 3.6 mmol) gave the title

(Aliph. C─H), 1735, 1636, 1575 (C N), 1459, 832–754 (C─Br).

8 of 11
EKIZ ET AL.
|
HRMS (ESI) m/z: calcd. for C16H11N2Br2 [M+H]+: 390.9269. Found:
129.2, 130.3, 131.0, 138.3, 139.6, 149.9, 153.7, 154.3. FT-IR (neat v∼
cm−1) 3384 (NH2), 3086, 2920–2849, 1673, 1585 (C N), 1465,

390.8980.
1317, 1123, 866–615 (C─Br). HRMS (ESI) m/z: calcd. for
10-Amine-2,8-dibromo-11-H-indeno[1,2-b]quinoline (15e)
2-Amino-5-bromobenzonitrile (13) (0.35 g, 1.78 mmol), 5-bromoin-
dan-1-one (14b) (0.38 g, 1.78 mmol), and InCl3 (0.47 g, 2.13 mmol)
gave the title compound (15e) (0.21 g, 30%). Light yellow powder solid,
mp 283–284°C. 1H NMR (300 MHz, DMSO-d6, ppm): δ 3.98 (s, 2H,
CH2), 7.80 (d, 4J = 2.8, 1H, ArH), 8.0–8.1 (m, 3H, ArH), 8.27 (d,
3J = 8.0 Hz, 1H, ArH), 8.78 (s, 1H, ArH), 8.85 (brs, ─NH), 9.04 (brs,
─NH). 13C NMR (75 MHz, DMSO-d6, ppm): δ 153.0, 151.5, 147.9,
137.6, 136.5, 133.3, 131.4, 129.6, 126.5, 126.1, 125.0, 122.3, 119.0,
117.6, 114.8, 33.8. FT-IR (neat v∼ cm−1) 3403–3318 (NH2), 3184
(Arom. C─H), 2923–2766 (Aliph. C─H), 1635, 1493, 1376, 1059,
862–741 (C─Br). HRMS (ESI) m/z: calcd. for C16H11N2Br2 [M+H]+:
390.9269. Found: 390.8984.
C
16H10N2Br [M+H]+: 468.8374. Found: 469.1268.
10-Amine-3,6,8-tribromo-11-H-indeno[1,2-b]quinoline (15i)
2-Amino-3,5-dibromobenzonitrile (12) (0.35 g, 1.27 mmol), 6-
bromoindan-1-one (14c) (0.27 g, 1.27 mmol), and InCl3 (0.31 g,
1.39 mmol) gave the title compound (15i) (0.09 g, 15% crude). Light
yellow powder solid, mp 291–292°C. 1H NMR (300 MHz, DMSO-d6,
ppm): δ 3.88 (s, 2H, CH2), 7.67 (d, 3J = 4.8 Hz, 1H), 7.74 (d, 3J = 4.8 Hz,
1H), 8.30 (d, 3J = 1.8 Hz, 1H), 8.28 (s, 1H), 8.42 (brs, 1H), 8.65 (brs, 1H),
8.73 (d, 3J = 4.2 Hz, 1H). 13C NMR (75 MHz, DMSO-d6, ppm): δ 153.0,
151.0, 144.7, 137.5, 136.5, 136.1, 134.5, 128.4, 126.5, 126.0, 122.3,
121.0, 119.1, 117.5, 115.4, 33.6. FT-IR (neat v∼ cm−1) 3312–3205

(NH2), 3131–3078, 2970, 1739, 1573 (C N), 1492, 1389, 1217,
1156, 875–734 (C─Br). HRMS (ESI) m/z: calcd. for C16H10N2Br
10-Amine-3,8-dibromo-11-H-indeno[1,2-b]quinoline (15f)
2-Amino-5-bromobenzonitrile (13) (0.35 g, 1.78 mmol), 6-bromoin-
dan-1-one (14c) (0.38 g, 1.78 mmol), and InCl3 (0.51 g, 2.30 mmol) gave
the title compound (15f) (0.16 g, 23%). Light brown powder solid, mp
280–281°C. 1H NMR (600 MHz, DMSO-d6, ppm): δ 3.89 (s, 2H, CH2),
7.70–7.84 (m, 2H, ArH), 7.92 (d, 3J = 9.3 Hz, 1H, ArH), 8.03 (dd,
3J = 9.0 Hz, 4J = 2.1 Hz,1H, ArH), 8.48 (s, 1H, ArH), 8.72 (s, 1H, ArH),
8.89 (brs, NH2) ppm. 13C NMR (150 MHz, DMSO-d6, ppm): δ 153.0,
151.0, 144.7, 137.5, 136.5, 136.1, 134.5, 128.4, 126.5, 126.0, 122.3,
121.0, 119.1, 117.5, 115.4, 33.6. FTIR (neat v∼ cm−1) 3556–3426
(NH2), 3198–3027 (Arom. C─H), 2967–2841 (Aliph. C─H), 1735,
[M+H]+: 468.8374. Found: 468.8329.
|
4.1.5 General procedures for the synthesis of
arylated indenoquinolines
To a solution of indenoquinoline (1.0 equiv) in 1,4-dioxane, aq Na2CO3
(3.0 M) 15 mL was added and stirred for 10 min at room temperature
under N2, followed by the addition of Pd(PPh3)4 (0.05 equiv) and
phenylboronic acid (1.3 equivalents for monobromides, 2.6 equivalents
for dibromides). The mixture was refluxed for 4 h at 90°C. The progress
of the reaction was monitored by TLC. After completion of the reaction,
the mixture was diluted with H2O and extracted with CHCl3 (3 × 20 mL).
The combined org. layers were dried (Na2SO4), filtered, and the filtrate
was concentrated in vacuo. The resultingresidue waspurified by column
chromatography (silica gel, CHCl3/MeOH).

1646, 1575 (C N), 1459, 831–734 (C─Br). HRMS (ESI) m/z: calcd.
for C16H11N2Br2 [M+H]+: 390.9269. Found: 390.8980.
10-Amine-1,6,8-tribromo-11-H-indeno[1,2-b]quinoline (15g)
2-Amino-3,5-dibromobenzonitrile (12) (0.35 g, 1.27 mmol), 4-bromoin-
dan-1-one (14a) (0.27 g, 1.27 mmol), and InCl3 (0.51 g, 2.30 mmol) gave
the title compound (15g) (0.12 g, 20%). Light yellow powder solid, mp
297–298°C. 1H NMR (300 MHz, DMSO-d6, ppm): δ 3.84 (s, 2H, CH2),
7.02(s,1H, ArH), 7.19(s,1H,ArH),7.49(t,4.6 Hz, 3J = 3.8, 1H, ArH),7.78
(d, 3J = 3.8 Hz, 1H, ArH), 8.32 (d, 3J = 4.6 Hz, 1H, ArH), 8.35 (brs, NH),
8.68 (brs, NH). 13C NMR (75 MHz, DMSO-d6, ppm): δ 155.7, 151.6,
148.3, 142.3, 141.2, 136.5, 133.5, 129.3, 128.7, 126.6, 124.8, 122.5,
121.5, 121.4, 32.7. FT-IR (neat v∼ cm−1) 3334–3225 (NH2), 3102–
10-Amine-1-phenyl-11-H-indeno[1,2-b]quinoline (16)
Starting with 15a (300 mg, 0.96 mmol), Pd(PPh3)4 (75.0 mg,
0.048 mmol), dioxane (15 ml), aq. K2CO3 solution (15 mL), and
PhB(OH)2 (150 mg, 1.25 mmol) were isolated as a brownish–yellow
crystalline solid 285 mg (96%), mp 234–235°C. 1H NMR (300 MHz,
CDCl3): 3.69 (s, 2H, CH2); 4.77 (brs, 2H, NH2); 7.33–7.73 (m, 10H,
ArH); 8.12 (d, 3J = 7.8 Hz, 1H, ArH); 8.24 (d, 3J = 6.6 Hz, 1H, ArH).
13C NMR (75 MHz, CDCl3): 31.6, 114.8, 118.2, 120.4, 121.1, 124.2,
127.6, 128.2, 128.7, 128.8, 129.0, 129.6, 130.0, 131.9, 132.2, 132.4,
139.4, 140.7, 142.0, 145.0, 149.4, 161.5. FT-IR (neat v∼ cm−1) 3389
(NH2), 3059 Arom. C─H), 2921–2850 (Aliph. C─H), 1633, 1567

3033 (Arom. C─H), 2970 (Aliph. C─H), 1656, 1574 (C N), 1402,
1377, 855–764 (C─Br). HRMS (ESI) m/z: calcd. for C16H10N2Br
[M+H]+: 468.8374. Found: 469.1268.

(C N), 1432, 1387. Anal. calcd. for C22H16N2: C, 85.69%, H,
10-Amine-2,6,8-tribromo-11-H-indeno[1,2-b]quinoline (15h)
2-Amino-3,5-dibromobenzonitrile (12) (0.83 g, 3 mmol), 5-bromoin-
dan-1-one (14b) (0.63 g, 3 mmol), and InCl3 (0.8 g, 3.6 mmol) gave the
title compound (15h) (0.08 g, 13%). Light yellow powder solid, mp
294–295°C. 1H NMR (300 MHz, CDCl3): δ 3.99 (s, 2H, CH2), 7.49 (d,
3J = 4.0 Hz, 1H, ArH), 7.55 (d, 3J = 3.9 Hz, 1H, ArH), 7.59–7.62 (m, 2H,
ArH), 7.70 (s, 2H, ArH), 7.71 (s, 1H, ArH) ppm. 13C NMR (75 MHz,
CDCl3, ppm): δ 32.6, 125.0 (2C), 125.4, 126.1, 126.9, 128.7, 129.1,
5.23%, N, 9.08%. Found: C, 85.72%, H, 5.25%, N, 9.03%.
10-Amine-2-phenyl-11-H-indeno[1,2-b]quinoline (17)
Starting with 15b (164 mg, 0.53 mmol), Pd(PPh3)4 (30.0 mg,
0.026 mmol), dioxane (15 ml), 3 M aq. K2CO3 solution (15 mL), and
PhB(OH)2 84.0 mg, 0.68 mmol) were isolated as a brownish–yellow
crystalline solid 155 mg (95%), mp 235–236°C. 1H NMR (300 MHz,
CDCl3-DMSO-d6): 3.93 (s, 2H, CH2); 6.00 (brs, 2H, NH2); 7.40–7.50 (m,

EKIZ ET AL.
9 of 11
|
4H, ArH); 7.60–7.72 (m, 4H, ArH); 7.85 (s, 1H, ArH); 8.03 (d, 3J = 8.5 Hz,
1H, ArH); 8.12 (d, 3J = 8.4 Hz, 1H, ArH); 8.23 (d, 3J = 8.0 Hz, 1H, ArH).
13C NMR (75 MHz, CDCl3-DMSO-d6): 32.0, 114.1, 118.1, 121.5,
121.7, 123.5, 124.0, 126.1, 127.1 (2C), 127.4, 128.6, 128.8 (2C), 129.0,
10-Amine-3,8-diphenyl-11-H-indeno[1,2-b]quinoline (21)
Starting with 15f (110 mg, 0.28 mmol), PhB(OH)2 (90 mg, 0.74 mmol)
and Pd(PPh3)4 (30 mg, 0.025 mmol) in dioxane (15 mL) and 3 M aq.
K2CO3 solution (10 mL), 21 was isolated as a brownish–yellow
crystalline solid (86 mg, 80%), mp 252–253°C. 1H NMR (300 MHz,
DMSO-d6, ppm): δ 3.83 (s, 2H, CH2); 7.32–7.76 (m, 15H, ArH); 8.09–
8.17 (m, 2H, ArH); 8.59 (s, 1H, ArH). 13C NMR (75 MHz, DMSO-d6,
ppm): δ 29.6, 115.0, 116.0, 121.4, 123.9, 126.4 (3C), 126.8, 127.1 (3C),
127.3 (3C), 127.4, 128.2 (3C), 129.0 (3C), 129.1, 129.2, 129.7, 140.6,
143.1, 145.6. FT-IR (neat v∼ cm−1) 3392 (NH2), 3068 (Arom. C─H),
140.6, 140.9, 141.8, 145.1, 146.3, 149.2, 160.7. FT-IR (neat v∼ cm−1
)
3451–3392 (NH2), 3058 Arom. C─H), 2922–2854 (Aliph. C─H),

1648, 1568 (C N). Anal. calcd. for C22H16N2: C, 85.69%, H,
5.23%, N, 9.08%. Found: C, 85.55%, H, 5.20%, N, 9.02%.
10-Amine-3-phenyl-11-H-indeno[1,2-b]quinoline (18)

Starting with 15c (76.0 mg, 0.24 mmol), Pd(PPh3)4 (14.0 mg,
0.012 mmol), dioxane (15 mL), 3 M aq. K2CO3 solution (15 mL), and
PhB(OH)2 (39.0 mg, 0.32 mmol) were isolated as a brownish–yellow
crystalline solid 68 mg (92%), mp 234–235°C. 1H NMR (300 MHz,
CDCl3): 3.82 (s, 2H, CH2); 4.76 (brs, 2H, NH2); 7.37–7.50 (m, 4H, ArH);
7.70–7.82 (m, 6H, ArH); 8.16 (d, 3J = 8.5, 1H, ArH); 8.49 (s, 1H, ArH).
13C NMR (75 MHz, CDCl3): 31.5, 115.3, 118.3, 120.2, 120.5, 124.5,
125.8, 127.6, 128.7 (3C), 129.0, 129.1 (2C), 129.9, 132.3, 140.8, 141.2,
142.1, 143.2, 145.0, 161.6. FT-IR (neat v∼ cm−1) 3803–3510 (NH2),
2922–2774 (Aliph. C─H), 1648, 1611, 1568 (C N), 1433, 1389,
965, 747. Anal. calcd. for C28H20N2: C, 87.47%, H, 5.24%, N,
7.29%. Found: C, 87.49%, H, 5.22%, N, 7.21%.
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4.2 Biology
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4.2.1 Anticholinesterase activity tests
Inhibitory activities of tested compounds against acetyl- and
butyrylcholinesterase were measured by slightly modified spectro-
photometric method developed by Ellman et al.[42] Acetylthiocholine
iodide and butyrylthiocholine iodide were used as substrates of the
reaction and DTNB method was used for the measurement of the
anticholinesterase activity. One hundred thirty microliters of 100 mM
sodium phosphate buffer (pH 8.0), test compound solutions and of
solution of AChE or BChE were mixed and incubated for 15 min at
25°C, and 0.5 mM DTNB was added. The reaction was then initiated by
the addition of acetylthiocholine iodide (0.71 mM) or butyrylthiocho-
line chloride (0.2 mM). The hydrolysis of these substrates was
monitored spectrophotometrically by the formation of yellow
5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB
with thiocholine, released by the enzymatic hydrolysis of acetyl-
thiocholine iodide or butyrylthiocholine chloride, at a wavelength of
412 nm. Acetylcholinesterase enzyme obtained from Electrophorus
electricus (Sigma–Aldrich, code: C3389) and butyrylcholinesterase
enzyme obtained from Equus caballus (Sigma–Aldrich, code: C7512)
were supplied from market. Tacrine and galanthamine were used as
standard compound. Ethanol was used as a solvent to dissolve test
compounds and the controls. The effect of ethanol excluded in the
calculation.

3128 Arom. C─H), 2924 (Aliph. C─H), 1645, 1565 (C N), 1506,
1433, 1363, 817, 758, 693. Anal. calcd. for C22H16N2: C, 85.69%,
H, 5.23%, N, 9.08%. Found: C, 85.65%, H, 5.22%, N, 9.15%.
10-Amine-1,8-diphenyl-11-H-indeno[1,2-b]quinoline (19)
Starting with 15d (30.0 mg, 0.077 mmol), PhB(OH)2 (24 mg, 0.2 mmol)
and Pd(PPh3)4 (9.0 mg, 0.008 mmol) in dioxane (15 mL) and 3 M aq.
K2CO3 solution (15 mL), 19 was isolated as a brownish–yellow
crystalline solid (25 mg, 83%), mp 251–252°C. 1H NMR (300 MHz,
DMSO-d6, ppm): δ 3.93 (s, 2H, CH2); 6.95 (brs, 2H, NH2); 7.36–7.38 (t,
J = 3.7, 1H, ArH); 7.43–7.58 (m, 7H, ArH); 7.68–7.70 (d, J = 3.8 Hz, 2H,
ArH); 7.88–7.90 (d, J = 3.8 Hz, 2H, ArH); 7.96 (brs, 2H, ArH); 8.05–8.06
(d, J = 3.7, 1H, ArH); 8.57 (s, 1H, ArH). 13C NMR (75 MHz, DMSO-d6,
ppm): δ 29.4, 114.0, 120.4, 120.6, 126.6, 127.3 (3C), 127.7, 127.9,
128.3, 129.0 (4C), 129.1 (4C), 129.3 (3C), 129.7, 139.3, 140.5, 142.5,
151.9, 162.6. FT-IR (neat v∼ cm−1) 3456–3322 (NH2), 3192, 3034

(Arom. C─H), 2921, 2770 (Aliph. C─H), 1639, 1558 (C N), 1491,
1385. Anal. calcd. for C28H20N2: C, 87.47%, H, 5.24%, N, 7.29%.
Found: C, 87.55%, H, 5.20%, N, 7.32%.
10-Amine-2,8-diphenyl-11-H-indeno[1,2-b]quinoline (20)
Starting with 15e (97.0 mg, 0.248 mmol), PhB(OH)2 (79 mg, 0.65 mmol)
and Pd(PPh3)4 (29.0 mg, 0.025 mmol) in dioxane (15 mL) and 3 M aq.
K2CO3 solution (15 mL), 20 was isolated as a brownish–yellow
crystalline solid (87 mg, 91%), mp 253–254°C. 1H NMR (300 MHz,
DMSO-d6, ppm): δ 3.93 (s, 2H, CH2); 7.05 (brs, 2H, NH2); 7.38–7.40 (m,
2H, ArH); 7.48–7.53 (m, 4H, ArH); 7.76–7.79 (m, 3H, ArH); 7.88–7.90 (d,
J = 3.9, 2H, ArH); 7.96 (brs, 2H, ArH); 8.09–8.11 (d, J = 3.9, 1H, ArH);
8.59 (s, 1H, ArH). 13C NMR (75 MHz, DMSO-d6, ppm): δ 29.5, 114.2,
118.5, 120.4, 121.9, 124.3, 126.3, 127.3 (4C), 127.4 (4C), 127.7, 128.0,
129.3 (4C), 129.4 (4C), 140.3, 140.7, 145.8. FT-IR (neat v∼ cm−1) 3346
(NH2), 3088 (Arom. C─H), 2919–2848 (Aliph. C─H), 1634, 1567
Percentage of inhibition of AChE or BChE was determined by a
comparison of the rates of reaction of samples relative to blank sample
(ethanol in phosphate buffer pH 8) using the formula:
½ðE ꢀ SÞ=Eꢁ ꢂ 100
where E is the activity of enzyme without test sample and S is the
activity of enzyme with test sample.
The anticholinesterase activity of 14 indenoquinoline amine
derivatives was measured at different concentrations (from
10−8 to 200 μM) for calculation of the IC50 values. Each substance
was tested three times in triplicate against AChE and BChE
enzymes.

(C N), 1489, 1386. Anal. calcd. for C28H20N2: C, 87.47%, H,
5.24%, N, 7.29%. Found: C, 87.40%, H, 5.22%, N, 7.34%.

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EKIZ ET AL.
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4.2.2 Carbonic anhydrase I and II isoenzymes
purification and inhibition studies
[12] M. Boztas, Y. Cetinkaya, M. Topal, I. Gu
̈
lc
̧
in, A. Menzek, E. Sahin, C. T.
Supuran, J. Med. Chem. 2014, 58(2), 640.
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Supuran, Bioorg. Med. Chem. 2015, 23(10), 2598.
The purification of both cytosolic CA isoforms (CA I and II) of human
blood cells was previously explained with a simple one-step procedure
by a Sepharose-4B-L-tyrosine-sulphanilamide affinity chromatoghra-
phy.[43–45] The protein quantity in the column effluents were evaluated
spectrophotometrically at 280 nm and previously described. For purity
of both isoenzymes sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) was applied. CA isoenzymes activities
were obtained in conforming with the procedure of Verpoorte et al.[46]
explained previously. The enhance in absorbance of the reaction
medium was spectrophotometrically obtained at 348 nm. We utilized
the esterase activity procedure for ascertaining the inhibition
agents by the Lineweaver–Burk procedure.[47] CA activity (%) versus
inhibitory concentration and 1/V versus 1/[S] graphs were drawn. The
quantity of protein was determined at 595 nm according to Bradford's
procedure.[48] Bovine serum albumin was used as standard protein as
given previously in detail. For the designation of the inhibition efficacy
of each novel indenoquinoline amine derivatives on both hCA
isoenzymes, an activity (%)-[indenoquinoline derivatives] graph was
drawn. The IC50 values were obtained from activity (%) versus
compounds plots. For the calculation of Ki values, substituted
indenoquinoline derivatives’ concentrations were used.
[14] U. M. Koçyiğit, Y. Budak, F. Eligüzel, P. Taslimi, D. Kılıç, İ. Gulc
̧in, M.
Ceylan, Arch. Pharm. 2017, 350, e1700198.
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e21995.
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Taslimi, İ. Gulçin, M. Ceylan, Arch. Physiol. Biochem. 2017, 124, 61.
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37, 896.
[18] S. Ökten, Ö. Y. Şahin, Ş. Tekin, O. Çakmak, J. Biotechnol. 2014, 185,
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[19] S. Ökten, O. Çakmak, Ş. Tekin, Turk. J. Clin. Lab. 2017, 8, 152.
[20] S. Ökten, O. Çakmak, Ş. Tekin, T. K. Köprülü, Lett. Drug Des. Dis. 2017,
14, 1415.
[21] R. Zong, D. Wang, R. Hammitt, R. P. Thummel, J. Org. Chem. 2006, 71,
167.
[22] M. Anzini, A. Cappelli, S. Vomero, A. Cagnotto, M. Skorupska, Med.
Chem. Res. 1993, 3, 249.
[23] M. Yamato, Y. Takeuchi, K. Hashigaki, Y. Ikeda, M. C. Chang,
K. Takeuchi, M. Matsushima, T. Tsuruo, T. Tashiro, S. Tsukagoshi,
Y. Yamashita, H. Nakano, J. Med. Chem. 1989, 32, 1295.
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Denny, Bioorg. Med. Chem. 2000, 8, 977.
[25] J. R. Brooks, C. Berman, M. Hichens, R. L. Primka, G. F. Reynolds, G. H.
Rasmusson, Proc. Soc. Exp. Biol. Med. 1982, 169, 67.
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[27] B. Venugopalan, C. P. Bapat, E. P. Desouza, N. J. Desouza, Indian J.
Chem. B 1992, 31, 35.
CONFLICT OF INTEREST
[28] A. Rampa, A. Bisi, F. Belluti, S. Gobbi, P. Valenti, V. Andrisano, V.
Cavrini, A. Cavalli, M. Recanatini, Bioorg. Med. Chem. 2000, 8, 497.
[29] S. Ökten, O. Çakmak, Tetrahedron Lett. 2015, 56, 5337.
[30] O. Çakmak, S. Ökten, Tetrahedron 2017, 73, 5389.
[31] M. Ekiz, A. Tutar, S. Ökten, Tetrahedron 2016, 72, 5323.
[32] O. Çakmak, R. Erenler, A. Tutar, N. Çelik, J. Org. Chem. 2006, 71,
1795.
The authors declare that they have no conflict of interest.
ORCID
Salih Ökten
http://orcid.org/0000-0001-9656-1803
http://orcid.org/0000-0001-8710-2912
http://orcid.org/0000-0002-3171-0633
Ümit M. Koçyiğit
Parham Taslimi
[33] A. Tutar, K. Berkil, R. R. Hark, M. Balcı, Synth. Commun. 2008, 38,
1333.
[34] R. Erenler, O. Çakmak, J. Chem. Res. 2004, 2004, 566.
[35] R. Erenler, I. Demirtaş, B. Buyukkidan, O. Çakmak, J. Chem. Res. 2006,
2006, 753.
[36] A. Şahin, O. Çakmak, I. Demirtas, S. Ökten, A. Tutar, Tetrahedron 2008,
64, 10068.
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How to cite this article: Ekiz M, Tutar A, Ökten S, et al.
Synthesis, characterization, and SAR of arylated
indenoquinoline-based cholinesterase and carbonic
SUPPORTING INFORMATION
anhydrase inhibitors. Arch Pharm Chem Life Sci. 2018;1–11.
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
https://doi.org/10.1002/ardp.201800167