Synthesis, characterization, UV–Vis absorption and cholinesterase inhibition properties of bis-indolyl imine ligand systems

Article abstract of DOI:10.1016/j.molstruc.2020.128308

A number of bis-indolyl imine helical structures has successfully been synthesized employing Schiff base reaction conditions starting from 4,6-dimethoxy-2,3-diphenylindole with different o-phenyl diamines as π-spacer bridged. The structures of targeted co

Full text of DOI:10.1016/j.molstruc.2020.128308

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Synthesis, characterization, UV–Vis absorption and cholinesterase inhibition
properties of bis-indolyl imine ligand systems
Mehmet F. Saglam, Murat Bingul, Elif Şenkuytu, Mehmet Boga, Yunus Zorlu, Hakan
Kandemir, Ibrahim F. Sengul
PII:
S0022-2860(20)30633-5
DOI:
https://doi.org/10.1016/j.molstruc.2020.128308
MOLSTR 128308
Reference:
To appear in: Journal of Molecular Structure
Received Date: 4 March 2020
Revised Date: 19 April 2020
Accepted Date: 20 April 2020
Please cite this article as: M.F. Saglam, M. Bingul, E. Şenkuytu, M. Boga, Y. Zorlu, H. Kandemir, I.F.
Sengul, Synthesis, characterization, UV–Vis absorption and cholinesterase inhibition properties of
bis-indolyl imine ligand systems, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/
j.molstruc.2020.128308.
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Mehmet F. Saglam: Investigation, Methodology, Writing- Original Draft
Murat Bingul: Investigation, Methodology, Writing- Original Draft
Elif Senkuytu: Investigation, Methodology, Writing- Original Draft
Mehmet Boga: Investigation, Validation
Yunus Zorlu: Software-Formal analysis, data curation, Writing- Original Draft
Hakan Kandemir: Investigation, Methodology, Validation
Ibrahim F. Sengul: Conceptualization, Methodology, Investigation, Writing-Review & Editing

Synthesis, Characterization, UV-Vis Absorption and Cholinesterase Inhibition
Properties of Bis-Indolyl Imine Ligand Systems
Mehmet F. Saglam1, Murat Bingul2, Elif Şenkuytu1, Mehmet Boga3, Yunus Zorlu1, Hakan
Kandemir4, Ibrahim F. Sengul1*
1Department of Chemistry, Faculty of Science, Gebze Technical University, Gebze, Kocaeli,
Turkey
2Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, Dicle University,
Diyarbakır, Turkey
3Department of Analytical Chemistry, Faculty of Pharmacy, Dicle University, Diyarbakır, Turkey
4Department of Chemistry, Faculty of Art and Science, Namık Kemal University, Tekirdag,
Turkey
*Correspondence: fazilsengul@gtu.edu.tr
Abstract
A number of bis-indolyl imine helical structures has successfully been synthesized employing
Schiff base reaction conditions starting from 4,6-dimethoxy-2,3-diphenylindole with different o-
phenyl diamines as π-spacer bridged. The structures of targeted compounds were identified by
1
FT-IR, mass, H and 13C NMR spectroscopy along with single crystal X-ray diffraction
techniques. The ground state absorption properties of the bis-indolyl compounds were also
investigated utilizing UV–Vis absorption spectroscopy. As the biological aspect of the
synthesized compounds, the anticholinesterase potency was investigated towards the
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. The highest inhibition
was determined in the presence of compound 9 with the values of 89.21 and 96.06, better than
standard Galantamine, for AChE and BChE, respectively.

Keywords: Indole, Schiff Base, UV-Vis, Cholinesterase Inhibitor
1. Introduction
Compounds containing heterocycles are some of the most important medical and industrial
molecules [1-4]. One such example is indoles backbones, which are an important class of
nitrogen containing heterocyclic compounds whose derivatives occur widely in many natural
products such as those from plants, fungi and marine organisms [5-7]. Over the past century,
intense study and considerable efforts have been devoted to the synthesis of indole and its
analogues [8-9]. As an emerging subclass, more complex indoles such as bis-indoles are a class
of indole alkaloids in which two indole motifs are connected to each other by different linkages
[10-12]. Bis-indole alkaloids are also an important structural class due to their interesting
biological properties and are therefore important target molecules for drug design studies [13-16].
Bis-indole motifs have been found to possess a diverse range of biological activities, for example
anti-tumor, anti-viral, anti-cancer, anti-inflammatory and antioxidant properties [15-22]. Bis-
indole analogues may also be used as acyclic or macrocyclic chelating ligand precursors and
serve as highly active and efficient metal complex catalysts for organic reactions [23-24].
Another significant feature of bis-indole scaffold in the field of supramolecular chemistry is that
these compounds are used as biologically and chemically important fluorescence probes because
of the acidic H-bond donor moiety of indoles [25-28].
The imine structure –N=CH-, commonly known as Schiff bases, is generally synthesized by the
condensation of a primary amine and an active carbonyl groups such as in aldehydes or ketones
with the elimination of water [29-30]. Schiff bases and their derivatives are an important class of
compound in organic chemistry, medicinal and supramolecular chemistry [29-31]. Schiff base
ligands are able to bind to metal ions via the availability of a donor lone pair on the nitrogen atom
to generate metal complexes with potential biological activities [32-33]. More specifically bis-
indole linked Schiff base containing compounds have a wide range of biological activities,
ranging from anti-bacterial to anti-cancer [34-37].
The importance of indole heterocyclic backbones for the design of new cholinesterase inhibitors
have been proved in literature [38-40]. The structural modification of Donepezil was carried out
by the use of indole moiety as bioisosteric substitute of the indanone moiety of Donepezil to
increase the potency of Donezepil through inhibition of AChE [41-42]. The tacrine, the first FDA

approved drug for the treatment of Alzheimer Disease, was also hybridized with the indole
moiety for the design of new multi-target directed AChE inhibitors [38].
Given the various potential applications of bis-indoles, it was of interest to synthesize bis-indoles
in which two indole units are connected with different o-phenylenediamines and investigate UV-
Vis properties. Moreover, AChE and BChE inhibition properties were also evaluated. To the best
of our knowledge, this is the first investigation of cholinesterase inhibition and photophysical
properties of methoxy activated 7,7'-diimine benzene linked bis-indole systems.
2. Experimental Section
2.1. Materials and methods
All reagents and solvents were obtained from commercial sources and appropriately purified, if
necessary. The deuterated solvent (CDCl3) for NMR spectroscopy was obtained from Merck.
Measurements IR spectra were recorded between 4000 and 650 cm-1 using a Perkin Elmer
Spectrum 100 FT-IR spectrometer. 1H and 13C NMR spectra were recorded in CDCl3 solution on
a Varian 500 MHz spectrophotometer. Matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF-MS) measurements were performed on a Bruker Daltonics
microTOF. Electronic absorption spectra were recorded with a Shimadzu 2101 UV
spectrophotometer in the UV-visible region.
2.2. X-ray data collection and structure refinement
Data for compounds 10 and 11 were obtained with Bruker APEX II QUAZAR three-circle
diffractometer. Indexing was performed using APEX2 [43]. Data integration and reduction were
carried out with SAINT [44]. Absorption correction was performed by multi-scan method
implemented in SADABS [45]. The structure was solved using SHELXT [46] and then refined
by full-matrix least-squares refinements on F2 using the SHELXL [47] in Olex2 Software
Package [48]. 4-Methyl-1,2-phenylene moiety (C1, C2, C3, C4, C5, C6, and C7) in compound 10
was disordered by symmetry over two configurations with occupancy 0.5. Chlorine atoms in 11
(Cl1 and Cl2) exhibited disorder over two sites with occupancies 0.75:0.25. Crystallographic data
and refinement details of the data collection for compounds 10 and 11 are given in Table 1.
Crystal structure validations and geometrical calculations were performed using Platon software
[49]. Mercury software was used for visualization of the cif files [50].

General Procedure: Synthesis of bis-indolyl Shiff-bases (9-12)
A reaction flask was charged with indole (2 mol equiv), diamine (1 mol equiv) and EtOH. To this
was added two drops of glacial acetic acid at room temperature. The reaction mixture was then
heated to reflux and stirred for 24-28 h. The reaction progress was monitored by TLC (ethyl
acetate:hexane, 50:50). Following the reaction complete, the reaction mixture was cooled to 0 οC
in an ice-bath for 15 min then precipitates were filtered, rinsed with hexane and dried under
vacuum to give bis-indolyl compound.
(1E,1'E)-N,N'-(1,2-Phenylene)bis(1-(4,6-dimethoxy-2,3-diphenyl-1H-indol-7-
yl)methanimine) (9)
This compound 9 was prepared from indole 4 (0.15 g, 0.42 mmol, 2 mol equiv), 1,2-
diaminobenzene 5 (23 mg, 1.6 mmol, 1 mol equiv), EtOH (9 mL) and 2 drops of acetic acid. The
yellow color suspension was stirred for 28 h under reflux. Following the workup according to the
general procedure gave the target product 9 (81 mg, 0.10 mmol) in 49% yield as yellow solid. Rf
1
0.64 (in EtOAc:hexane (50:50)); H NMR (500 MHz, CDCl3)
δ 11.79 (bs, 2H, indole NH), 9.26
(s, 2H, imine CHN), 7.33–7.29 (m, 14H, Ar-H), 6.85 (t, J = 7.3 Hz, 2H, Ar-H), 6.80 (d, J = 7.6
Hz, 4H, Ar-H), 6.72 (t, J = 7.3 Hz, 4H, Ar-H), 6.22 (s, 2H, Ar-H5), 3.99 (s, 6H, OCH3), 3.80 (s,
6H, OCH3) ppm; 13C NMR (125 MHz, CDCl3)
δ 158.8 (CHN), 156.2, 146.4, 136.7, 136.3, 132.9,
132.2, 131.5, 127.7, 127.3, 127.2, 126.2, 125.8, 119.6, 114.1, 102.9, 87.8 (Ar-C), 56.91 (OCH3),
55.42 (OCH3) ppm; IR (ATR-thin film) νmax = 3300, 1614, 1590, 1557, 1464, 1366, 1244, 1219
cm–1; MALDI-TOF (m/z) calc for C52H42N4O4: 786.321; found: 786.057.
(1E,1'E)-N,N'-(4-Methyl-1,2-phenylene)bis(1-(4,6-dimethoxy-2,3-diphenyl-1H-indol-7-
yl)methanimine) (10)
This compound 10 was prepared from indole 4 (0.10 g, 0.28 mmol, 2 mol equiv), 4-methyl-o-
phenylenediamine 6 (17 mg, 0.14 mmol, 1 mol equiv), EtOH (5 mL) and 1 drop of acetic acid.
The browny color suspension turned to orange color under reflux and this suspension was stirred
for 24 h under reflux. Following the workup according to the general procedure gave the target
product 10 (81 mg, 0.10 mmol) in 72% yield as orange solid. Rf 0.60 (in EtOAc:hexane (50:50));
1H NMR (500 MHz, CDCl3)
δ 11.79 (bs, 2H, indole NH), 9.22 (s, 2H, imine CHN), 7.35–7.20
(m, 10H, Ar-H), 7.10 (s, 2H), 6.87–6.74 (m, 6H), 6.73–6.64 (m, 4H), 6.20 (s, 2H), 3.97 (s, 3H,
13
OCH3), 3.96 (s, 3H, OCH3), 3.77 (s, 6H, OCH3), 2.47 (s, 3H, CH3) ppm; C NMR (125 MHz,

CDCl3)
128.6, 128.0, 127.6, 127.3, 127.1, 126.2, 125.8, 120.3, 119.6, 119.3, 118.1, 116.0, 114.1, 113.4,
102.9, 87.9 (Ar-C), 56.9 (OCH3), 55.4 (OCH3), 21.2 (CH3) ppm; IR (ATR-thin film) νmax
δ 155.9 (CHN), 155.6, 146.1, 143.9, 136.7, 136.4, 136.0, 132.9, 132.2, 131.5, 131.4,
=
3359, 3259, 2982, 1618, 1598, 1546, 1514, 1465, 1364, 1250, 1217 cm–1; MALDI-TOF (m/z)
calc for C53H44N4O4: 800.336; found: 800.051.
(1E,1'E)-N,N'-(4-Chloro-1,2-phenylene)bis(1-(4,6-dimethoxy-2,3-diphenyl-1H-indol-7-
yl)methanimine) (11)
This compound 11 was prepared from indole 4 (0.15 g, 0.42 mmol, 2 mol equiv), 4-chloro-o-
phenylenediamine 7 (30 mg, 0.21 mmol, 1 mol equiv), EtOH (9 mL) and 2 drops of acetic acid.
The red color suspension turned to brown color during reflux and this suspension was stirred for
28 h under reflux. Following the workup according to the general procedure gave the target
product 11 (97 mg, 0.12 mmol) in 56% yield as yellow solid. An analytical sample of 11 was
1
obtained by recrystallization from CH2Cl2/hexane. Rf 0.67 (in EtOAc:hexane (50:50)); H NMR
(500 MHz, CDCl3)
δ 11.67 (s, 1H, indole NH), 11.63 (s, 1H, indole NH), 9.21 (s, 2H, imine
CHN), 7.27–7.17 (m, 13H, Ar-H), 6.83 (t, J = 7.1 Hz, 2H, Ar-H), 6.80–6.73 (m, 4H, Ar-H),
6.73–6.64 (m, 4H, Ar-H), 6.21–6.17 (m, 2H, Ar-H5), 3.99 (s, 3H, OCH3), 3.96 (s, 3H, OCH3),
3.79 (s, 2H, OCH3), 3.78 (s, 3H, OCH3) ppm; 13C NMR (125 MHz, CDCl3)
δ 156.8 (CHN),
156.4, 137.7, 136.7, 136.2, 132.9, 132.2, 131.5, 131.1, 127.7, 127.3, 127.2, 126.2, 125.9, 125.7,
120.5, 119.5, 87.7 (Ar-C), 56.8 (OCH3), 55.4 (OCH3) ppm; IR (ATR-thin film) νmax = 3300,
2933, 1589, 1541, 1463, 1366, 1242, 1218 cm–1; MALDI-TOF (m/z) calc for C52H41ClN4O4:
820.282; found: 820.118.
(1E,1'E)-N,N'-(4-Nitro-1,2-phenylene)bis(1-(4,6-dimethoxy-2,3-diphenyl-1H-indol-7-
yl)methanimine) (12)
This compound 12 was prepared from indole 4 (0.15 g, 0.42 mmol, 2 mol equiv), 4-nitro-o-
phenylenediamine 8 (32 mg, 0.21 mmol, 1 mol equiv), EtOH (9 mL) and 2 drops of acetic acid.
The orange color reaction mixture was stirred for 28 h under reflux. Following the workup
according to the general procedure gave the target product 12 (79 mg, 0.095 mmol) in 45% yield
as red solid. Rf 0.67 (in EtOAc:hexane (50:50)); 1H NMR (500 MHz, CDCl3) δ 11.55 (s, 1H,
indole NH), 11.15 (s, 1H, indole NH), 9.24 (s, 1H, imine CHN), 9.20 (s, 1H, imine CHN), 8.17
(s, 1H, Ar-H), 8.00 (s, 2H, Ar-H), 7.47–7.25 (m, 16H, Ar-H), 6.77–6.67 (m, 4H, Ar-H), 6.29–
6.19 (m, 2H, Ar-H), 4.01 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.81 (s, 3H,

OCH3) ppm; 13C NMR (125 MHz, CDCl3) δ 160.39, 160.13, 159.86, 157.93, 157.63, 152.42,
147.29, 146.74, 145.58, 136.68, 132.71, 131.44, 128.66, 127.73, 127.64, 127.59, 127.36, 127.19,
127.12, 127.07, 114.51, 114.22, 112.83, 101.98, 87.48 (Ar-C), 56.65, 55.43 (OCH3) ppm; IR
(ATR-thin film) νmax = 3503, 3400, 1595, 1561, 1544, 1491, 1450, 1363, 1322, 1284, 1249, 1121
cm–1; MALDI-TOF (m/z) calc for C52H41N5O6: 831.306; found: 831.232.
2.3. Biological studies
Anticholinesterase activity determination method
AChE and BChE inhibitory activities were measured by the slightly modified spectrophotometric
method reported in the literature [51]. Galanthamine was used as the standard.
% Inhibition = (A0 – A1) / A0 x 100
where A0 is the absorbance of the control and A1 is the absorbance in the presence of the sample.
Statistical analysis
The results were mean ± SD of three parallel measurements. All statistical comparisons were
made by means of Student’s t-test, p values < 0.05 were regarded as significant.
3. RESULTS AND DISCUSSION
3.1. Synthesis and Characterization
In general, the most reactive site of the indole nucleus with respect to electrophilic substitution is
the C3 position [10-11]. In the event that C3 is blocked by a substituent, electrophilic substitution
is diverted to the C2 position [12]. The reactivity at the C7 position can be increased by the two-
electron donating methoxy groups at the C4 and C6 positions [14]. 2,3-Diphenyl-4,6-dimethoxy
indole was the parent scaffold for this study. This indole has its active C2 and C3 positions
blocked with phenyl groups leaving the C7 position as the only active site.
The preparation of bis-indolyl imine systems required the synthesis of 4,6-dimethoxy-2,3-
diphenylindole 3 as a starting material which was synthesized in high yield by the one-pot
reaction of commercially available 3,5-dimethoxyaniline with benzoin. After refluxing overnight
in glacial acetic acid, the compound was obtained as white solid [52]. Since the C7 position of the
targeted indole ring in the molecule 3 is especially susceptible to electrophilic aromatic
substitution, it was selected for a further functionalization at C7 position in order to construct 7,7-

bis-indole structures. Aldehydes functional groups are well-known precursors to imine formation
through Schiff base reaction. Regardingly, Black et al., have reported that 4,6-dimethoxy-2,3-
diphenylindole 3 can be directly formylated using the Vilsmeier-Haack reagents phosphoryl
chloride and DMF to yield the related indole-7-carbaldehyde 4 in a high yield (Scheme 1) [53].
Scheme 1. Reagents and conditions: a) CH3COOH, aniline, 130 ºC; b) DMF, POCl3, 0 ºC.
The next requirement for successful Schiff base synthesis was the condensation of the carbonyl
group with primary o-phenylenediamines. For this purpose, treatment of compound 4 with
commercially available o-phenylenediamine 5 in absolute ethanol for 6h under reflux was found
to give successfully the corresponding indole-based imine macrocyclic compound 9 in moderate
yield as a yellow powder. Similar treatment of the compound 4 with 4-methyl, chloro, and nitro
substitute o-phenylenediamines 6-8 gave the corresponding 7,7-linked bis-indolyl imine
structures 10-12 in reasonable yields (Scheme 2).
Scheme 2. Reagents and conditions: a) EtOH, AcOH, reflux.
The solid-state structures of compounds 10 and 11 were solved using single-crystal X-ray
analysis in order to get a better insight into the molecular conformation (Fig. 1). Suitable crystals
for X-ray diffraction study of compounds 10 and 11 were grown via recrystallization from

dichloromethane-hexane solvent mixture. As shown in Fig. 1 A-B and C-D, X-ray analysis
exhibited that π-spacer (o-phenylene) bridged dimers containing indole rings favored a helical
form in the solid state, which stabilized by the intra-molecular hydrogen bonding interactions
(H1···N2 = 2.185 Å in compound 10 and H4···N3 = 2.256 Å, H1···N2 = 2.218 Å in compound
11). This helical orientation in compounds 10 and 11 are very similar to compounds derived from
the π-spacer-bridged dimers [54].

Figure 1. Perspective views of crystal structures of compounds 10 (top) and 11 (bottom) showing
the intra-molecular CH···N hydrogen bonding interactions. Indole rings are shown in light
yellow.
Table 1. Crystal data and refinement parameters for compounds 10 and 11.
1997697
C53H44N4O4
800.92
1985535
C52H41ClN4O4
821.34
CCDC
Empirical Formula
Formula weight (g. mol-1)
Temperature (K)
Radiation
296
300
MoKα (λ = 0.71073) MoKα (λ = 0.71073)
Monoclinic
C2/c
Triclinic
P-1
Crystal system
Space group
18.821(3)
14.485(2)
16.806(4)
90
12.134 (7)
13.759 (6)
14.373 (7)
82.74 (3)
77.018 (18)
75.748 (16)
0.06 × 0.07 × 0.17
2260.0 (19)
2
a
b
c
(Å)
(Å)
(Å)
α
(°)
92.695(14)
90
β
(°)
γ
(°)
Crystal size (mm)
0.23× 0.24 × 0.28
4576.5(14)
4
V
(Å3)
Z
ρcalcd (g. cm−3)
1.162
1.207
µ (mm−1)
0.074
0.13
1688
860
F
(000)
4.334 to 50.054
5.834 to 49.996
2θ range for data collection (°)
h/
k/l
-22 ≤ h ≤ 22, -15 ≤ k -14 ≤ h ≤ 14, -16 ≤ k
≤ 17, -19 ≤ l ≤ 19
≤ 16, -17 ≤ l ≤ 16
12060
15321
Reflections collected
Independent reflections
4026 [Rint = 0.0653, 7804 [Rint = 0.1435,
Rsigma = 0.0787] Rsigma = 0.2835]

4026/45/296
1.06
7804/0/565
0.82
Data/restraints/parameters
Goodness-of-fit on
2 (S)
2 > 2 2)]
wR(F2)
F
0.0749
0.091
R
[F
σ(F
0.2379
0.221
1
The H NMR and 13C NMR spectra of compound 10 were characteristic for compounds 9-12.
1
The H NMR spectrum of compound 10 showed presence of a broad singlet at 11.79 ppm
corresponding to the NH protons and the downfield of this resonance is due to the formation of
H-bond between imine nitrogen and indole NH groups. The twelve methoxy protons on the
1
benzene ring appeared as three sets of sharp singlets at 3.77, 3.96 and 3.97 ppm. The H NMR
spectrum of compound 10 also showed the replacement of the aldehyde protons at 10.43 ppm by
the new imine protons at 9.22 ppm. In a similar fashion, 13C NMR spectrum supported this
13
structure, indicating the loss of two CHO carbons as a result of imine formation. The C NMR
spectrum also indicated methyl carbon on the 4-methyl o-diphenyl diimine at 21.2 ppm and two
methoxy carbon attached to indole ring system at 55.4 and 56.9 ppm. The IR spectra of the
compound 10 displayed absorption at 1618 cm-1 corresponding to the imine stretching frequency.
In addition, the exact molecular weight of the compounds 9-12 were determined by MALDI-TOF
mass spectrometry, which revealed the anticipated molecular ions at m/z 786, 800, 820, 831
(M+1) respectively.
3.2. Ground State Electronic Absorption Properties of Compound 9-12.
The ground state absorption properties of bis-indolyl imine systems (9-12) were recorded in
common organic solvent including tetrahydrofuran, dioxane, dichloromethane, chloroform,
methanol, ethanol, acetonitrile, dimethylsulfoxide and N,N-dimethylformamide at room
temperature (Fig. 3a, S17-S19). The absorption spectra of the targeted compounds 9, 10, 11 and
12 in different solvents were compared. Although the absorbance intensity of the compounds
with the solvent effect changed, it was observed that there was no significant change in the
absorbance bands. The absorbance bands observed at 290-490 nm for 9-12 in all studied solvents
can be attributed to π-π* transitions [55-57]. The maximum absorption band for all compounds in
dimethylsulfoxide was observed as broadband at 410, 340, 340 and 400 nm, respectively (Fig. 2).
The ground state absorption spectra of the compounds were also obtained at different
concentrations and the molar extinction coefficients were calculated according to the Beer-

Lambert law (Fig. 3b, S20-S22). Molar extinction coefficients (ε) of the of compounds 9-12
were calculated as 4.29x104 M-1 cm-1 (for 410 nm), 4.05x104 M-1 cm-1 (for 340 nm), 0.50x104 M-
1 cm-1 (for 340 nm), and 0.55x104 M-1 cm-1 (for 400 nm), respectively.
1
0.8
9
10
0.6
11
0.4
12
0.2
0
250
300
350
400
450
500
550
600
Wavelength(nm)
Figure 2. Absorbance spectra of 9-12 (10 µM) in dimethylsulfoxide.

Figure 3. (a) Absorbance spectra of 9 (10 µM) in different solvents; (b) Absorbance spectrum of
9 in DMSO at different concentration.
3.3. Determination of Anticholinesterase Activity
The novel bis-indole imine systems 9-12 were screened to determine their anticholinesterase
activity using acetylcholine and butyrylcholine enzyme inhibition assays. The inhibition of AChE
and BChE by the compounds is given as percentage values at 200 µM concentrations in Table 2

and compared with the galantamine as a standard. The compounds showing higher percentage of
inhibitions were evaluated as potent candidates for the treatment of Alzheimer Disease. As
expected, the positive control galantamine, showed very potent inhibition both on AChE and
BChE with values of 78.76% and 79.72%, respectively.
Table 2. Acetylcholinesterase and butyrylcholinesterase enzyme inhibitory activities of 9-12.a,b
Sample
AChE (% Inhibition)
89.21±1.39
BChE (% Inhibition)
96.06±1.41
NA
9
10
11
12
30.48±0.97
48.76±1.28
NA
38.76±0.67
31.73±0.24
79.72±0.33
Galantamineb
78.76±0.94
a Values expressed are means ±S.D. of three parallel measurements. (p < 0.05)
b 200 µM
c Positive control
In general, the better inhibition values were obtained in the case of AChE and the best candidate
was determined with no substitution on the phenyl ring, compound 9, administered by the
aromatic diamine. The inhibition values of 89.21% and 96.06% for both AChE and BChE by the
treatment of compound 9 resulted better inhibition potency than the galantamine used as
standard. The replacement of hydrogen atom on 4 position of o-phenylenediamine with electron
withdrawing groups Cl and NO2 decreased the inhibition potencies in both AChE and BChE. The
compound 12 containing nitro substitution displayed moderate inhibitions towards the AChE and
BChE with the values of 38.76% and 31.73%, while the compound 11, with chloro substituent
was determined as active for AChE and not applicable inhibition was detected for the BChE. The
electron donating methyl substituent on the aromatic imine linker 10 dramatically decreased the
inhibition potency towards the designated enzymes. The inhibition value was determined as
30.48% in the case of AChE, while no inhibition was found for the BChE.
The results obtained from the biological studies could reasonably be explained by the possible
binding patterns between the designated compounds and different binding sites on the AChE and
BChE. It is known that, a range of different active locations namely anionic, esteratic and acyl
binding sites are found on the catalytic side of AChE and BChE. [58-60]. On this purpose, the

main consideration for the chosen of indole moiety as the structural backbone of the targeted
compounds was the creation of hydrophobic interactions due to the presence of aromatic rings
with the anionic sites of the enzymes. It was thought that, the addition of different substituents
could be valuable to understand if there might be further hydrophobic and/or hydrogen bond
interactions with the abovementioned binding sites. The methyl residue could create new
hydrophobic binding with the anionic sites while the electron withdrawing Cl and NO2 groups
could form hydrogen bonds with esteratic binding sites. However, according to the results, none
of the proposed interactions were observed and the unsubstitued bis-indolyl imines compound 9
was detected as the most potent compound. It was postulated that the presence of any residue on
the aromatic ring administered by the phenyl diamine destroyed the optimal interactions between
the compound and the binding sites. It was also assumed that there was no responsible
aminoacids on the catalytic side of the enzymes to create additional interactions with the electron
donating and withdrawing substituents.
4. Conclusion
A range of methoxy activated indole containing cyclic tetradentate ligand systems has been
successfully synthesized employing well-known Schiff base reactions conditions. Schiff base
reaction of indole-7-carbaldehyde with o-phenylene diamines provide a convenient approach to
the synthesis of larger tetradentate open chain compounds. All bis-indolyl imine structures were
elucidated by NMR, mass, IR experiments and X-ray crystal structures have been obtained for
compounds 10 and 11. Preliminary biological assays indicated that the synthesized bis-indolyl
imine systems have a moderate impact on inhibition of AChE and BChE enzymes. One further
point to be noted is that this class of bis-indole tetradentate structure could be considered as a
potential molecular receptor for neutral small molecules.
Acknowledgments
This work has been supported by Research Found of the Gebze Technical University (Project
Number: 2019-A-105-37). We thank to Dr. Michael Dieter Wörle for his support in refining
compound 10.
Appendix A. Supplementary material
Supplementary data were given as Supporting Information. Additional crystallographic data with
CCDC reference numbers 1997697 (for 10) and 1951435 (for 11) have been deposited within the
Cambridge Crystallographic Data Center via www.ccdc.cam.ac.uk/deposit. These data can be

obtained free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif
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•
•
•
•
•
Synthesis and characterizations of Bis-indolyl tetradentate ligand systems are presented.
Single crystals were grown, and their structures were analyzed.
Uv-Vis absorptions are investigated.
Cholinesterase inhibition properties were investigated toward AChe and BChe enzymes.
Compound 9 exhibited excellent potency for AChe and BChe as compared to reference
Galatamine.

There is no declaration of Interest