Synthesis, characterization and crystal structure of new tetrahydro-β-carboline as acetylcholinesterase inhibitor

Article abstract of DOI:10.1016/j.molstruc.2019.127070

In this study, a new tetrahydro-β-carboline derivative, 2-benzoyl-6-methoxy-9-methyl-1-phenyl-1,2,3,4-tetrahydro-β-carboline has been synthesized through a three-steps reaction in good yield (76%). The structure of this compound was characterized by different spectroscopic methods including NMR (1D and 2D), UV, IR and MS. Molecular structure of the synthesized compound was also confirmed by single crystal X-ray diffraction technique. Further, evaluation of in vitro acetylcholinesterase (AChE) inhibitory activity showcased the potential of this compound as AChE inhibitor with an IC₅₀ value of 26.52 ± 0.79 μM. In addition, this compound showed minimal toxicological profile at cellular level. Docking simulation illustrated a highly selective binding at peripheral side of AChE via hydrophobic interactions. In silico prediction of blood-brain barrier (BBB) permeation and ADMET properties of the compound was also reported.

Full text of DOI:10.1016/j.molstruc.2019.127070

Journal Pre-proof
Synthesis, characterization and crystal structure of new tetrahydro-β-carboline as
acetylcholinesterase inhibitor
Ahmad Saifuddin Mohamad Arshad, Nelson Jeng Yeou Chear, Mohammed Oday
Ezzat, Nur Aziah Hanapi, Ramu Meesala, Suhana Arshad, Sharif Mahsufi Mansor,
Mohd Nizam Mordi
PII:
S0022-2860(19)31170-6
DOI:
https://doi.org/10.1016/j.molstruc.2019.127070
MOLSTR 127070
Reference:
To appear in: Journal of Molecular Structure
Received Date: 15 May 2019
Revised Date: 10 September 2019
Accepted Date: 11 September 2019
Please cite this article as: A.S. Mohamad Arshad, N.J. Yeou Chear, M.O. Ezzat, N.A. Hanapi, R.
Meesala, S. Arshad, S.M. Mansor, M.N. Mordi, Synthesis, characterization and crystal structure of new
tetrahydro-β-carboline as acetylcholinesterase inhibitor, Journal of Molecular Structure (2019), doi:
https://doi.org/10.1016/j.molstruc.2019.127070.
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X-ray crystallographic
analysis
In silico BBB permeation
AChE inhibition
IC50 = 26.52 ± 0.79 µM
and ADMET
Cytotoxicity study
Binding mode analysis

Synthesis, characterization and crystal structure of new tetrahydro-
β-carboline as acetylcholinesterase inhibitor
Ahmad Saifuddin Mohamad Arshada*, Nelson Jeng Yeou Cheara, Mohammed
Oday Ezzatb, Nur Aziah Hanapia, Ramu Meesalac, Suhana Arshadd, Sharif Mahsufi
Mansora and Mohd Nizam Mordia
a Centre for Drug Research, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
b Department of Chemistry, College of Education for Women, University of Anbar, 31001
Ramadi, Anbar, Iraq
c Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur,
Malaysia
d X-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, 11800 Minden,
Penang, Malaysia
*Corresponding author: E-mail: saifudin_pudin@yahoo.com; Fax: +604-6568669; Tel.: +604-
6533888 Ext. 2145.
In this study, a new tetrahydro-β-carboline derivative, 2-benzoyl-6-methoxy-9-methyl-1-phenyl-
1,2,3,4-tetrahydro-β-carboline has been synthesized through a three-steps reaction in good yield
(76%). The structure of this compound was characterized by different spectroscopic methods
including NMR (1D and 2D), UV, IR and MS. Molecular structure of the synthesized compound
was also confirmed by single crystal X-ray diffraction technique. Further, evaluation of in vitro
acetylcholinesterase (AChE) inhibitory activity showcased the potential of this compound as
AChE inhibitor with an IC50 value of 26.52 ± 0.79 µM. In addition, this compound showed
minimal toxicological profile at cellular level. Docking simulation illustrated a highly selective
binding at peripheral side of AChE via hydrophobic interactions. In silico prediction of blood-
brain barrier (BBB) permeation and ADMET properties of the compound was also reported.
Keywords: Tetrahydro-β-carboline; Single crystal; Acetylcholinesterase; Molecular docking;
Cytotoxicity; ADMET
1

1. Introduction
Alzheimer’s disease (AD) is the progressive neurodegenerative disorder and the leading
cause of dementia commonly affecting the elderly population. It is associated with the decrease
of cognitive functions, thinking skills and understandings, which are related to memory function
and ability to perform daily routines [1-2]. A number of hypotheses have been proposed in the
pathogenesis of AD, with the most widely accepted are acetylcholine (ACh) imbalance, amyloid
beta (Aβ) production and its aggregation as well as tau hyperphosphorylation [3]. To date, there
is no breakthrough drug candidate or a single pathway that can treat the underlying cause of AD.
Available drugs provide symptomatic improvement alone but do less to modify the disease
process. Nonetheless, various therapeutic strategies have been discovered to slow down its
progression by targeting the disease modification part [2]. Among these strategies are, inhibition
of acetylcholinesterase (AChE), N-methyl-D-aspartate (NMDA) receptor, monoamine oxidase
(MAO), amyloid precursor protein (APP) and secretase enzymes [2, 4].
Cholinergic system, which plays an important role in the regulation of learning,
cognition, and memory processes, has been extensively studied for the design of anti-
Alzheimer’s drugs [5]. The so-called cholinergic hypothesis states the deficiency of
neurotransmitter ACh in the human brain is a crucial factor associated with AD pathogenesis
which contributed to its cognitive symptoms [6]. This theory has led to the development of
acetylcholinesterase inhibitors (AChEIs) that are able to restore cholinergic function by blocking
the AChE, consequently increasing the synaptic levels of ACh in the brain [7]. To date, the use
of AChEIs is the most prevalent effective AD symptomatic treatment. In fact, the first-line
treatment that is prescribed to AD patients is AChEIs [8]. Till date, four AChEIs approved by the
2

U.S Food and Drug Administration (FDA) for the symptomatic treatment of AD are donepenzil,
rivastigmine, galantamine and tacrine [9]. However, tacrine is no longer routinely prescribed due
to its high incidence of hepatotoxicity reported at therapeutic doses [10]. So far, the only licensed
drug that has a different mode of action from AChEIs is memantine, which acting as a NMDA
receptor antagonist [11].
To date, a number of highly active AChEIs had been discovered, including synthetic,
semi-synthetic and natural compounds [12]. While structurally diverse, these AChEIs are
primarily alkaloids [13-14]. Most notably, all marketed drugs currently being licensed to
alleviate cognitive symptoms in dementia are from the class of alkaloids. From a long time, the
indole alkaloids containing 1,2,3,4-tetrahydro-β-carboline (THBC) skeleton have represented
important lead structures in view of their wide range of biological activities [15]. Its core
structure or sub-unit appears in a large number of biologically active compounds and present in
drugs currently available on the market as well as drug candidates under development. While
there are many findings reflecting the potential of its unsaturated member, β-carbolines (BCs) as
anti-AChE agents [16-17], data on the anti-AChE activity of THBCs are relatively sparse and
inconclusive, possibly due to poor activities reported [18-19]. However, based on the limited
number of THBCs tested, this would lead to erroneous conclusions on their ineffectiveness in
treating AD. Additionally, it is important to note that the toxicity issue of BCs remains as the
major drawback for it to be developed into therapeutic drug [20-21]. Taking into account these
limitations, we prompted to synthesis and evaluate new THBC derivative for anti-AChE activity
using various in silico and in vitro models.
3

2. Experimental
2.1 Materials and reagents
All the chemicals and solvents were commercially available and used as purchased
without further purification. The reaction progress was monitored using thin-layer
chromatography (TLC). TLC was performed on aluminium-backed silica gel plates, Kieselgel 60
F254 (Merck, Germany) with spots visualized under UV light.
2.2 Physical measurements
13
1D (1H and C) and 2D (DEPT 90, DEPT 135, COSY, HSQC, HMBC) NMR spectra
were recorded in deuterated chloroform (CDCl3) using Bruker AV 500 MHz Ultrashield NMR
1
spectrometer. The chemical shifts are reported in ppm downfield to TMS (δ = 0) for H NMR
and relative to the central CDCl3 resonance (δ = 77.0) for 13C NMR. Spin multiplicities are
expressed as s (singlet), d (doublet), t (triplet) and m (multiplet) while coupling constants (J) are
given in Hertz. FT-IR spectrum was recorded on a Nicolet 6700 FT-IR spectrometer (Thermo
Scientific, MA, USA) in the mid-IR region (400-4000 cm-1) using Attenuated Total Reflection
(ATR) technique. The UV-vis spectrum was recorded in MeOH as the solvent using UV-1800
UV-Visible Recording Spectrophotometer (Shimadzu Cooperation, Kyoto, Japan) in the range of
200-800 nm at room temperature in a quartz cuvette (path length = 1 cm). The mass spectrum
was recorded on an Agilent 6890 N Network GC system coupled to an Agilent 5973i Mass
Selective Detector.
4

2.3 Synthesis
2.3.1 Preparation of intermediates 1 and 2
Intermediate 1 was synthesized in good yield according to the published method using 5-
methoxytryptamine as the starting material in presence of trifluoroacetic acid (TFA) and
benzaldehyde [22]. Intermediate 2 was synthesized according to the published method with
slight modifications using triethylamine (TEA) in refluxing toluene [23].
2.3.2 Preparation of 2-benzoyl-6-methoxy-9-methyl-1-phenyl-1,2,3,4-tetrahydro-β-carboline (3)
A mixture of intermediate 2 (0.382 g, 1 mmol, 1equiv) and dimethylformamide (5mL)
was stirred at room temperature for 10 minutes, and then 60% sodium hydride (2 mmol) and
methyl iodide (1.5 mmol) were added. The mixture was stirred at room temperature. After
completion of reaction as indicated by TLC, the resulting mixture was poured into water and
extracted with ethyl acetate. The organic layer was collected, washed with water and brine, then
dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The
resulting oil was purified by column chromatography using a mixture of hexane and ethyl acetate
as an eluent, recrystallized using methanol:chloroform (1:1) solvent system to give compound 3
as block-like brown crystals. Yield (76%), UV (λmax/nm): 280.5, 225.5; IR ATR (vmax/cm-1): 2918
2
3
(aromatic Csp –H stretching), 2855 (alkyl Csp –H stretching), 1624 (carbonyl C=O stretching);
1H NMR (δ/ppm, 500 MHz, CDCl3): δ 7.32-7.24 (10 aromatic protons, H-12 to16 and H-18 to
22), 7.30 (H-8, 1H, d, J = 8.5 Hz), 7.08 (H-1, 1H, s), 6.92 (H-5, 1H, bd, J = 2.0 Hz), 6.83 (H-7,
1H, dd, J = 8.5, 2.0 Hz), 3.79 (O-CH3, 3H), 3.33 (N-CH3, 3H), 3.65 (H-3β, 1H, dd, J = 13.5, 6
Hz), 3.24 (H-3α, 1H, td, J = 27.0, 13.5, 4.0 Hz), 2.84 (H-4β, 1H, td, J = 27, 15, 5.5 Hz); 2.67 (H-
4α, 1H, dd, J = 15.5, 4.0 Hz) (Methylenes, H-4α / H-4β and H-3α /H-3β are interchangeable,
5

13
respectively). C NMR (δ/ppm, 125 MHz, CDCl3): δ 169.45 (C-10), 153.06 (C-6), 138.48 (C-
17), 135.38 (C-11), 132.35 (C-9b), 131.61 (C-9a), 128.54 (C-4b), 127.74 – 125.45 (aromatic
carbons, C-12 to 16; C-18 to 22),110.52(C-7),108.79 (C-8), 106.91 (C-4a), 99.45(C-5), 55.00 (O-
CH3, C-23), 50.26(C-1), 40.08(C-3), 28.88(C-9), 21.31 (C-4). EI-MS (70 eV) m/z: 396.
Scheme 1. Synthesis of the tetrahydro-β-carboline derivatives 1-3. Reagents and reaction
conditions: (i) 10% TFA/PhCHO; (ii) PhCOCl, TEA, reflux, 24 h; (iii) MeI, NaH, DMF.
2.3.3 Crystallization
A
supersaturated solution was prepared by dissolving compound
3
in
methanol:chloroform mixture (1:1) at ambient temperature. The prepared solution was slightly
warmed and allowed to evaporate slowly at room temperature. After about one week, good
quality transparent crystals appeared were allowed to grow to a maximum possible dimension
and then harvested. The single crystal obtained was used for X-ray diffraction studies.
2.4 Single crystal X-ray diffraction studies
X-ray diffraction data for compound 3 was collected from single crystal by using a
Bruker APEX II Duo CCD diffractometer with a graphite monochromatic Mo-Kα radiation (λ=
0.71073 Å) with APEX2 software [24]. The collected data had been reduced by using the SAINT
program [24] and the empirical absorption corrections were performed with the SADABS
program [24]. The crystal structure was solved by direct methods and was refined using the full-
6

matrix least-squares method on F2 using the SHELXTL program [25-26]. All geometrical
calculations were carried out using the program PLATON [27]. The molecular graphics were
drawn using SHELXTL [25] programs. All non-hydrogen atoms were refined anisotropically.
All the hydrogen atoms bound to C atoms were positioned geometrically [C-H = 0.93-0.97 Å]
and refined using riding model, with isotropic displacement parameters set to 1.2(C) and
1.5(Cmethyl) times the equivalent isotropic U values of the parent carbon atoms. A rotating group
model was applied to the methyl groups. The final refinement converged well. Details of the data
collections condition and the parameters of the refinement process are given in Table 1.
Table 1. Crystal data and structure refinement for the title compound
CCDC
1891690
Formula
C26H24N2O2
396.47
294
0.71073
Monoclinic
P21/n
Formula Weight (gmꢀmole-1)
Temperature (K)
Wavelength (Å)
Crystal System
Space group
Unit cell (Å, °)
a = 9.6916(8), b = 23.9861(19), c = 9.8782(8)
β = 116. 4237(14)
2056.4(3)
Volume (Å3)
Z
4
Calculated density (gꢀcm-3)
Absorption coefficient (mm-1)
F(000)
1.281
0.081
840
Crystal size (mm)
Theta min – max (°)
Dataset
0.20 x 0.47 x 0.60
2.4 – 32.6
-14≤ h ≤ 14; -36≤ k ≤ 36; -14≤ l ≤ 14
28416, 7478, 0.035
4879
Total/unique data/ R(int)
Observed data [I>2.0σ(I)]
Data/parameters
7478, 273
0.0567, 0.1779, 1.03
R[a]/wR2[b]/Goodness-of-fit[c]
Largest diff. peak and hole eꢀÅ-3 -0.22, 0.31
w=1/[σ2(Fo ) + (0.09252+ 0.1838P] (I), where P = (Fo + 2Fc2)/3.[a]R=Σ||Fo| – |Fc||/Σ|Fo|.[b]Rw={wΣ(|Fo| –
2
2
|Fc|)2/Σw|Fo|2}}1/2 [c]
.
GOF={Σw(|Fo| – |Fc|)2/(n–p)}1/2, where n is the number of reflections and p is the total number
of parameters refined.
7

2.5 In vitro acetylcholinesterase enzyme inhibitory assay
The AChE inhibitory potential was evaluated using a modified Ellman’s method [28].
Initially, 140 ꢁL of 0.1 M sodium phosphate buffer (pH 8) was first added to a 96-well
microplate, followed by the addition of 20 ꢁL of compounds and 20 ꢁL of 0.09 units/mL AChE.
After 15 minutes of incubation at 25 °C, 10 ꢁL of 10 mM 5,5′-dithiobis-2-nitrobenzoic acid
(DTNB) was added into each well followed by 10 ꢁL of 14 mM acetylthiocholine iodide. 30
minutes after the initiation of an enzymatic reaction, the absorbance of the colored end-product
was measured using Thermo ScientificTM MultiskanTM GO Microplate Spectrophotometer at
412 nm. A set of five concentrations of each compound was used to estimate the 50% inhibitory
concentration (IC50). Galantamine, a cholinesterase inhibitor, was used as the reference standard.
The absorbance of the compounds was corrected by subtracting the absorbance of their
respective blank. Percentage of inhibition was calculated using the following formula:
Inhibition (%) = (Abscontrol – Abssample)/(Abscontrol) × 100
2.6 Cell culture works
2.6.1 Cell line and culture condition
Hepatocellular carcinoma (Hep G2 cell line) and colorectal adenocarcinoma (HT-29 cell
line) were obtained from American Type Culture Collection (ATCC). Hep G2 cells were
cultured in Eagle’s Minimum Essential Medium (EMEM) while HT-29 cells were cultured in
McCoy’s 5a Medium Modified. Both cultures were supplemented with 10% fetal bovine serum
(FBS) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin solution). These cells
were maintained as adherent culture in a humidified atmosphere of 5% CO2 incubator at 37 °C.
8

2.6.2 Preparation of cell culturePTED MANUSCRIPT
Initially, cells were allowed to grow under optimal incubator conditions. Subculturing of
the cells was performed after the cells reached 70-80% confluence. The old medium was first
aspirated and the cells were washed twice with pre-warmed sterile phosphate buffered saline
(PBS) and then completely discarded. A solution of 1 mL trypsin-EDTA was then added and
distributed evenly onto the surface of the cells in 75 cm2 cell culture flask. The cells were placed
in an incubator at 37 ºC in 5% CO2 atmosphere for 5 min. To facilitate cells segregation, the
flask containing the cells was gently tapped. Immediately after detachment of cells, trypsin
activity was deactivated by adding 2 mL of fresh complete medium (10% FBS). The medium
containing cells were then transferred to a sterile centrifuge tube and upon centrifugation, the
supernatant was discarded. The cell pellet was resuspended with medium and counted with the
aid of haemacytometer. Cells were then subcultured into new tissue culture flask containing fresh
media and incubated in a humidified atmosphere of 5% CO2 at 37 °C.
2.6.3 MTT assay
The cells were harvested and approximately 1x104 cells in 100 µL media were seeded in
a sterile 96-well plate and incubated overnight. After a recovery period of 24 hours, different
concentrations of compounds were added into wells and the plate was then incubated for 48
hours at 37 °C with 5% CO2 in a humidified atmosphere. Cells treatment was performed in
triplicates for each concentration. 20 µL of MTT was then added to each well followed by 3
hours of incubation period in the dark environment. The medium was aspirated out and 100 µL
of DMSO was added to dissolve the formazan crystals formed. Finally, the absorbance was taken
at 570 nm using Thermo ScientificTM MultiskanTM GO Microplate Spectrophotometer. Cisplatin
was used as the positive control and the negative control was represented by the untreated
9

medium containing DMSO. Absorbance of the compounds was corrected by subtracting the
absorbance of their respective blank. Percentage of inhibition was calculated using the following
formula:
Inhibition (%) = (Abscontrol – Abssample)/(Abscontrol) x 100
2.7 Molecular docking studies with AChE enzyme
The X-ray crystal structure of enzyme AChE (PDB: 1EVE, resolved at 2.5 Å) complexed
with the ligand donepezil was retrieved from protein data bank for structure-based anti-
Alzheimer drug design [29]. Using Glide version 5.7 (Schrödinger LLC, New York, USA), the
most active compound (3) was docked onto the active site of refined AChE structure. Prior to
docking, AChE crystal structure was prepared and refined. Firstly, water molecules and hetero
groups were deleted from receptor beyond the radius of 5 Å of reference ligand, and the resulting
protein structure was refined and minimized by Protein Preparation Wizard using OPLS-2005
force field. Based on site points of the complexed ligand, an active site of protein was defined
and confirmed from literature. Receptor Grid Generation program was used to prepare AChE
grid. Ligands were optimized by LigPrep program using OPLS-2005 force field to generate the
lowest energy state of respective ligands. Docking simulations were handed in 5 poses per
ligand, in which the best pose with the highest score was displayed for each ligand. The docking
protocol was validated by a reproduction of the binding pose of donepezil in 1EVE. Further, the
protocol was substantiated by docking reported AChEIs within the active site. The best-ranked
pose of ligands was studied to determine their binding as well as interactions within the AChE
active site.
10

2.8 In silico BBB permeation and ADMET studies
In silico BBB permeation and ADMET studies were carried out with QikProp version 3.3
(Schrodinger LLC, New York, USA) software running on Windows 7, 64 bit using a workstation
computer, with an Intel Corei7 6X2.6 Gigahertz processor and 16 Gigabyte memory. The
designed structure was optimized by LigPrep using OPLS-2005 force field. Out file of LigPrep
was used as an input file for QikProp to determine various pharmacokinetic descriptors. QikProp
jobs were monitored and results were obtained in the form of OUT file.
3. Results and Discussion
3.1 Synthesis
The synthetic pathway leading to the title compound is depicted in Scheme 1. In the
initial step, 5-methoxytryptamine was cyclized using Pictet-Spengler reaction in presence of
benzaldehyde and 10% catalytic amount of trifluoroacetic acid to afford tricyclic tetrahydro-β-
carboline intermediate 1. In the subsequent step, synthesis of intermediate 2 was accomplished
by N2-benzoylation by heating intermediate 1 with benzoyl chloride in presence of triethylamine
in refluxing toluene. In the final step, N9-methylation of intermediate 2 at room temperature
produced the desired compound in good yield. The structure was well elucidated using various
spectroscopic techniques before its cholinesterase activity was investigated.
3.2 Characterization
In the IR spectrum of compound 3 (Figure S1, supplementary file), the diagnostic
2
infrared absorption bands can be assigned to the aromatic Csp –H stretching (2918 cm-1), alkyl
3
Csp –H stretching (2855 cm-1) and carbonyl C=O stretching (1624 cm-1). Most of the bands as
observed in the IR spectrum of compound 3 suggest the existence of all the functional groups
present in the compound. The UV-Vis absorption spectrum of the compound 3 (Figure S2,
11

supplementary file) showed two absorption peaks at 225.5 nm and 280.5 nm. The presence of a
strong UV absorption peak at 225.5 nm is due to the π-π* transition of the aromatic ring system
on the chromophore structure. On the other hand, a weak and broad peak at 280.5 nm is due to
the n-π* transition, mainly involves the transition of a non-bonding (lone pair) electron on the
oxygen up to a π* antibonding molecular orbital. There was no absorption peak observed
between 320 nm to 800 nm in the entire visible region. The shape of the absorption spectrum for
compound 3 is found to be almost identical to its core tetrahydro-beta-carboline scaffold.
1
In the H-NMR spectrum (Figure S3, supplementary file), the presence of trisubstituted
aromatic protons, a sharp peak of N-CH3 and one CH2-CH2-N- group were observed, indicating
the compound has a tetrahydro-β-carboline skeleton (ring A, B and C) [30-31]. The 1,3,4-
trisubstituted aromatic protons in ring A appeared as a doublet of doublets at δH 6.83, a doublet
at δH 7.30 and a broad doublet at δH 6.92 attributable to H-7 (1H, dd, J = 8.5, 2.0 Hz), H-8 (1H,
d, J = 8.5 Hz) and H-5 (1H, bd, J = 2.0 Hz), respectively. The sharp singlet at δH 3.33 (3H) was
identified as the N-methyl proton resonance of B-ring. Two sets of methylene protons in ring C
were found at δH 2.67 (1H, dd, J = 15.5, 4.0 Hz, H-4α), 2.84 (1H, td, J = 27, 15, 5.5 Hz, H-4β)
and δH 3.65 (1H, dd, J = 13.5, 6 Hz, H-3β), 3.24 (1H, td, J = 27, 13.5, 4 Hz, H-3α), respectively.
The COSY spectrum of 3 as shown in (Figure S4, supplementary file), revealed a clear
correlation between H-7 (δH 6.83) and H-8 (δH 7.30). A cross-correlation between H-3 and H-4
was also observed in the COSY spectrum. On the other hand, the singlet at δH 7.08 was a
characteristic proton (H-1) of the tetrahydro-β-carboline C ring.
12

The 13C NMR spectrum (Figure S5, supplementary file) showed the presence of 26
carbons, corresponding to its molecular formula, C26H24N2O2. With the help of DEPT 135 and
90 (Figure S6-S7, supplementary file), the carbons were further divided into 2 methyls, 2
methylenes, 13 methines, and 8 quaternary carbons. Utilizing the HSQC data (Fig. 1), the signals
at δc99.45, 110.52 and 108.79 were assigned to C-5, C-7 and C-8 based on their direct
correlations with the protons on the A-ring of tetrahydro-β-carboline skeleton. The quaternary
carbon, C-6 was assigned to the signal at δc153.06 due to its correlation with H-5 (δH6.92), H-7
(δH 6.83) and H-8 (δH 7.13) in the HMBC experiment (Fig. 2). The typical methoxy singlet, δH
3.79 (3H, s) was then found to have a long range correlation (HMBC) with the carbon signal at δ
153.06 ppm (C-6), thus confirming the methoxy group was substituted at 6 position of the ring
A. The quaternary carbons, C-4a, C-4b, C-9a and C-9b of ring B were assigned to the signals at
δc106.91, 128.54, 131.61 and 132.35 ppm, respectively. The assignment of C-4a at signal δc
106.91 was evident by its correlations with H-3β (δH3.65), H-4α (δH 2.67), H-4β (δH 2.84), H-1
(δH 7.08) and H-5 (δH 6.92) in the HMBC experiment. C-4b was assigned to δc 128.54 due to its
HMBC correlations to H-8 and H-1. The quaternary carbon, C-9a was identified with the signal
at δc131.61 since it exhibited long range correlations to the aromatic protons, H-5, H-7, H-8 of
the A-ring. C-9b was assigned to δc132.35 due to its correlations with H-1 and H-4 (C-ring) in
HMBC. The position of C-9a and C-9b were further confirmed by their HMBC cross signals
with the sharp N-CH3 singlet (δH 3.33) at 9-position of the B-ring.
13

Fig. 1. 1H-13C HSQC spectrum of compound 3 (CDCl3, 500 MHz).
Fig. 2. 1H-13C HMBC spectrum of compound 3 (CDCl3, 500 MHz).
14

The ketone carbon, C-10 was assigned to the far most downfield signal, δc169.45. The
assignment of C-10 was further substantiated by its long range correlations with H-3α, H-3β and
H-1 of ring C in HMBC. Two sets of overlapping signals (10 H) with identical splitting patterns
were observed between δH 7.24 and 7.32, corresponding to two mono-substituted aromatic units.
Two benzylic quaternary carbons, C-11 and C-17 were found at δc 135.38 and 138.48,
respectively. The assignment of C-17 at δ 138.48 was evident by its HMBC correlations with H-
1 of ring C, suggesting that the first aromatic ring substituted at position 1 of the C-ring. Lastly,
C-11 was assigned to δc 135.38, however, the HMBC data was found to be too weak for any
indication. Nevertheless, based on what was discovered later from the crystallographic data, it is
confirmed that the second aromatic ring substituted at C-10, the carbonyl chain of ring C. All
1
1
possible long range correlations of H-13C HMBC and H-1H COSY correlations are shown in
Fig. 3. In addition, the structure and the stereochemistry of compound 3 were confirmed by the
single crystal X-ray crystallographic analysis.
Fig. 3. 1H-13C HMBC and 1H-1H COSY correlations of compound 3.
3.3 X-ray diffraction (XRD) studies
The ORTEP diagram of the molecular structure of the title compound (C26H24N2O2) with
assigned atom-numbering scheme is shown in Fig. 4. Table 2 lists the selected bond distances
and angles. All bond lengths and angles are comparable with the previous closely related
15

structures [32-34]. From the crystallographic data, the block-like brown crystal of the title
compound was crystallized in P21/c space group with a = 9.6916 (8)Å, b = 23.9861 (19)Å, c =
9.8782 (8)Å, β = 116.4237 (14)°, V = 2056.4 (3) Å3 and Z = 4. X-ray structure analysis
confirmed the absolute configuration of the stereogenic center in the title compound as S-
configuration. In the molecular structure, the tetrahydropyridine ring (N2/C7-C11) within the β-
carboline fused ring system adopts a half-chair conformation [35] where C8–C9 bond is twisted
with puckering parameters of Q= 0.4654(15) Å and φ= 45.6(2)°. Similar conformations are also
observed from the previous β-carboline reported structures [32-34]. Additionally, the methoxy
substituent group is also found to be slightly twisted with C13—O1—C4—C3 torsion angles
being -167.62(14)°, showing that methoxy groups are oriented in antiperiplanar
conformations.The dihedral angle between the two terminal benzene rings (C15-C20 and C21-
C26) is 82.18(8)°, further demonstrates the coplanar structure of the title compound.
Fig. 4. The molecular structure of the title compound, showing 50% probability displacement ellipsoids and the
atom numbering scheme.
16

Table 2 Selected bond distances, bond and torsion angles for the title compound.
Atoms
Bond Distances(Å)
O1— C4
O1— C13
1.3781(16)
1.405(2)
O2— C14
1.227(2)
N1—C12
1.452(2)
N2—C9
N2—C10
N2—C14
C14—C15
1.4642(19)
1.4783(19)
1.3537(19)
1.502(2)
Bonds Angles (°)
C4—O1—C13
C1—N1—C12
C11—N1—C12
C9—N2—C10
C9—N2—C14
117.65(13)
124.93(12)
127.10(11)
116.88(11)
124.70(13)
123.31(11)
122.40(16)
119.78(14)
117.73(13)
N1—C11—C10
O2—C14—N2
O2—C14—C15
N2—C14—C15
Torsion Angles (°)
C13—O1—C4—C3
C12—N1—C1—C2
C12—N1—C11—C10
N2—C10—C11—N1
C21—C10—C11—N1
C9—N2—C10—C11
C14—N2—C10—C11
C9—N2—C14—O2
C10—N2—C14—O2
C9—N2—C14—C15
C10—N2—C14—C15
O2—C14—C15—C20
-167.62(14)
6.1(2)
-6.4(2)
173.72(11)
-63.16(16)
38.35(14)
-141.76(11)
-170.60(14)
9.5(2)
13.02(19)
-166.88(11)
-110.08(17)
The crystal structure of the title compound is shown in Fig. 5 and the hydrogen bond
geometry is listed in Table 3. The molecules are connected by C3—H3A…Cg2 [symmetry code:
x,y,1+z and C3…Cg2 distance:3.6299(18)Å] and C24—H24A…Cg1[symmetry code: 1-x,-y,-z and
C24…Cg1 distance:3.7077(18)Å] interactions where Cg1 and Cg2 represent the centroids of C1-
C6 and C21-C26 aromatic rings, respectively. These C—H…π interactions link the molecules
into infinite one dimensional column along the c-axis.
17

Fig. 5. The crystal packing of the title compound, showing the C—H…π interactions.The H atoms not involved in
the intermolecular interactions have been omitted for clarity.
Table 3 Hydrogen bond geometry.
D—H(Å)
0.95
D—H…A
D—H…A (°)
130
H…A (Å)
2.96
D…A (Å)
3.6299(18)
3.7077(18)
C3—H3A…Cg2
C24—H24A…Cg1
0.95
2.99
135
Symmetry code (i) x,y,1+z; (ii) 1-x,-y,-z; Cg1 and Cg2 are the centroids of C1-C6 and C21-C26 benzene rings,
respectively.
3.4 Acetylcholinesterase inhibitory activity and molecular docking
The title compound and intermediates were evaluated for their cholinesterase inhibitory
activity against acetylcholinesterase (AChE) enzyme using in vitro cholinesterase inhibition
assays (Ellman's assay method). The results are summarized in Table 4. Although cholinesterase
activity of THBC compounds are not comparable with the standard drug galantamine (IC50 of
0.94 ± 0.17 µM), all compounds showed moderate activity with IC50 values in the range of
26.52-89.05 µM. Intermediates 1 and 2 showed the anti-AChE activity with IC50 values of 89.05
18

and 62.86 µM, respectively. Interestingly, compound 3 incorporated of indole-N-methyl
substituent showed a 2-fold increase in its anti-AChE activity.
Table 4 The acetylcholinesterase inhibitory activity of compounds 1-3.
Compounds
1
2
3
IC50 values (µM)
89.05 ± 5.21
62.86 ± 3.05
26.52 ± 0.79
0.94 ± 0.17
Galantamine
In an attempt to rationalize the observed in vitro enzymatic activity as well as investigate
the possible binding mode of the most active compound (3), docking study into the active site of
AChE was performed. For results comparison, galantamine (currently used as a drug for
Alzheimer’s disease) was docked as a positive control. Compound 3 showed quite high binding
affinity (high negative binding score -4.998), even higher than that of the chosen standard (Table
5). The docking analysis reveals that compound 3 interact with AChE enzyme primarily due to
the hydrophobic interactions. Inside AChE active site, compound 3 showed a highly selective
binding at the peripheral anionic site of AChE involving hydrophobic interactions with TYR70,
TYR121, TRP279, PHE290 and TYR334 (Fig. 6). Notably, these highly selective hydrophobic
interactions were also observed when galantamine was docked into the active site of AChE, thus
referring to the ability of this compound to superimpose in the same position of active drug.
Table 5 Docking score and close contact residues (surrounding amino acids within 4 Å of ligand)
Compound
Docking score (kcal/mol)
Surrounding amino acids within 4
Å of ligand
-4.998
TYR70, TYR121, ASP276,
TRP279, ARG289, PHE290,
TYR334, GLY335
3
Galantamine
-2.732
TYR70, ILE275, ASP276,
TRP279, ASN280, TYR334
19

Fig. 6. Binding modes of compound 3 with AChE: (Left) Hydrophobic contacts of 3 (ball and sticks) with AChE
residues (ribbon) within 4Å cut-off distance. (Right) Embedment of compound 3 (ball and sticks) into peripheral
anionic site of AChE surface.
3.5 Cytotoxicity evaluation
To address the toxicity issue, all compounds were tested for their potential cytotoxicity
against HepG2 as well as HT-29 cells using MTT assay. The IC50 values of more than 100 µM
shown in Table 6 for both cell lines after 48 hours of incubation period showcased its low
cytotoxicity. All compounds were considered non-toxic within tested concentration range of
3.125 to 100 µM as more than 80% of the cells were viable at all concentrations [36]. This non-
toxic profile might due to the loss of planarity in THBC skeleton as shown by the crystal
structure. In contrast, its unsaturated congener of fully aromatized β-carboline has planar
conformation and potent cytotoxicity owing to their ability to intercalate into DNA double helix,
which can further cause cell apoptosis [37-38]. These results demonstrated preliminary safety as
both HepG2 and HT-29 cell lines are utilized as in vitro liver and intestinal models for toxicity
testing, respectively [39].
20

Table 6 Cytotoxicity studies of compounds 1-3
Compounds
IC50 values (µM)
HT-29
Hep G2
>100
>100
1
>100
>100
2
>100
>100
3
Cisplatin
14.01±1.73
28.37±3.81
3.6 In silico BBB permeation and ADMET studies
The ability to permeate across the blood-brain barrier (BBB) is essential for drugs acting
on the central nervous system (CNS) like cholinesterase inhibitors. It is determined by their
molecular size and physicochemical properties, as well as by complex binding and elimination
processes occurring in the circulation, peripheral organs, at the BBB and in the brain parenchyma
and its fluids [40]. Estimated QikProp descriptors for in silico prediction of BBB permeability of
compound 3 (Table 7) were within the range of values for 95% of known drugs (according to
Schrödinger software, https://www.schrodinger.com/qikprop). ADME results for CNS parameter
seem to be favourable and indicate that the desired compound can be targeted for CNS activity.
Notably, compound 3 had values of three descriptors (QPlogBB, QPPMDCK and QPPCaco)
which can reflect directly to the ability of a molecule to permeate the BBB falling into the
recommended range [41-42]. The predicted value for BBB penetration (QPlogBB) indicating
brain/blood partition coefficient of compound 3 was in the optimum range (-3 to 1.2).
Additionally, the result of QPPMDCK parameter describing the permeability of MDCK cells
mimic blood–brain barrier was in the prescribed range. Further, another data from surrogate
BBB model utilizing the permeability of Caco-2 cells (QPPCaco) used as a model for gut–blood
barrier was in the permissible range [42].
The effect of lipophilicity on BBB permeability (QPlogPo/w) is another principle
descriptor important for CNS penetration. Most CNS-acting agents are lipophilic and can cross
21

the BBB by passive diffusion [43]. The QPlogPo/w value of compound 3 was at the higher range
of the recommended values, that indicates good passive diffusion. Moreover, compound 3 gave
the lowest polar surface area (PSA) value, which was below the recommended threshold of 90
Å2, which also favors possible passive BBB permeation [44]. In fact, PSA value equal to or less
than 140 Å2 is predicted to have good oral bioavailability, which is in agreement with the
predicted percent human oral absorption (100%) [45].
In accordance with Lipinski’s rule of five (Ro5) for the prediction of drug-likeness and
orally bioavailability in humans [46], desired compound was small in size with molecular weight
less than 400 g/mol, absence of hydrogen bond donors (HBD), hydrogen bond acceptors (HBD)
less than four and fewer number of rotatable bonds (ROTB). In fact, CNS penetration requires a
sum of the heteroatoms (O + N) of 5 or less [47] with the marketed CNS drugs on average have
number of these heteroatoms = 4.32 [48]. This is close to the number of these heteroatoms
present in compound 3 (4), indicating its potential for CNS penetration. Additionally, the
presence of tertiary nitrogen in desired structure (a feature of many CNS drugs) shows a higher
degree of brain permeation [49]. Overall, in silico ADME profiles and prediction of BBB
permeation indicate that compound 3 can be thought to be CNS active and has the ability to cross
the BBB endothelium by passive permeation. Additionally, the descriptor values were found to
be in the QikProp specified ranges, so this compound seems to possess favourable
pharmacokinetic properties [50].
Table 7 In silico BBB permeation and ADMET profiles of compound 3
Descriptors
3
CNS (-2 to +2)
MW < 500 Da
HBD (0-6)
1
396.488
0
HBA (2-20)
Qplog Po/w (-2.0 – 6.5)
ROTB (0-15)
3.75
5.783
2
22

QPlog BB (-3.0 – 1.2)
0.226
QPPMDCK < 25 poor > 500 good
QPPCaco < 25 poor > 500 good
PSA (7-200 Å)
3468.582
6060.539
35.084
% Human oral absorption < 25% poor > 80% good 100
#N and O
Violation of Ro5 (max 4)
4
1
4. Conclusion
A new tetrahydro-beta-carboline derivative was successfully synthesized and its chemical
structure was characterized by physical and spectroscopy techniques. In addition, the structure
and the stereochemistry of the compound were confirmed by the single crystal X-ray
crystallographic analysis. The compound was found to be a moderate AChE inhibitor with
relatively low cytotoxicity. The observed inhibitory activity was further rationalized using
molecular docking simulation. Notably, this compound has well predicted BBB permeation and
bioavailability. Taken together, these findings indicate that this compound could be further
modified to achieve better potency, and a potential candidate for Alzheimer’s disease therapeutic
agents.
Acknowledgements
This work was supported by the Research University (RUT) Grant Scheme
(1001/CDADAH/855005)
and
Research
University
(RUI)
Grant
Scheme
(1001/CDADAH/8011075), Universiti Sains Malaysia, Malaysia. This work was partly funded
by the Ministry of Higher Education of Malaysia under the HICoE Programme
(311/CDADAH/4401009). Ahmad Saifuddin Mohamad Arshad and Nelson Jeng Yeou Chear
thank Institute of Postgraduate Studies, USM for providing USM Fellowship Scheme for
financial support.
23

References
[1] S. Sheikh, Safia, E. Haque, S.S. Mir, Neurodegenerative diseases: Multifactorial
conformational diseases and their therapeutic interventions, J. Neurodegener. Dis. 20 (2013) 1–8.
[2] R. Anand, K.D. Gill, A.A. Mahdi, Therapeutics of Alzheimer’s disease: Past, present and
future, Neuropharmacology. 76 (2014) 27–50.
[3] X. Du, X. Wang, M. Geng, Alzheimer’s disease hypothesis and related therapies, Transl.
Neurodegener. 7 (2018) 2.
[4] E. Scarpini, P. Schelterns, H. Feldman, Treatment of Alzheimer’s disease: Current status and
new perspectives, Lancet Neurol. 2(9) (2003) 539–547.
[5] T.H. Ferreira-Vieira, I.M. Guimaraes, F.R. Silva, F.M. Ribeiro. Alzheimer’s disease:
Targeting the cholinergic system, Curr. Neuropharmacol. 14(1) (2016) 101–115.
[6] L.A. Craig, N.S. Hong, R.J. McDonald, Revisiting the cholinergic hypothesis in the
development of Alzheimer's disease, Neurosci. Biobehav. Rev. 35(6) (2011) 1397-1409.
[7] H. Wang, H. Zhang, Reconsideration of anticholinesterase therapeutic strategies against
Alzheimer’s disease, ACS Chem. Neurosci. 10(2) (2019) 852-862.
[8] J. Birks, Cholinesterase inhibitors for Alzheimer’s disease, Cochrane Database Syst. Rev. 1
(2006) CD005593.
[9] A. Lleo, Current therapeutic options for Alzheimer’s disease, Curr. Genomics. 8(8) (2007)
550–558.
[10] P.B. Watkins, H.J. Zimmerman, M.J. Knapp, S.I. Gracon, K.W. Lewis, Hepatotoxic effects
of tacrine administration in patients with Alzheimer’s disease, JAMA. 271(13) (1994) 992-998.
[11] S.J. Thomas, G.T. Grossberg, Memantine: A review of studies into its safety and efficacy in
treating Alzheimer’s disease and other dementias, Clin. Interv. Aging. 4 (2009) 367-377.
[12] M. Singh, M. Kaur, H. Kukreja, R. Chugh, O. Silakari, D. Singh, Acetylcholinesterase
inhibitors as Alzheimer therapy: From nerve toxins to neuroprotection, Eur. J. Med. Chem. 70
(2013) 165-188.
[13] P.J. Houghton, Y. Ren, M.J. Howes, Acetylcholinesterase inhibitors from plants and fungi,
Nat. Prod. Rep. 23(2) (2006) 181-199.
[14] P. Williams, A. Sorribas, M-J.R. Howes, Natural products as a source of Alzheimer’s drug
leads, Nat. Prod. Rep. 28(1) (2011) 48–77.
[15] A.E. Laine, C. Lood, A.M. Koskinen, Pharmacological importance of optically active
24

tetrahydro-β-carbolines and synthetic approaches to create the C1 stereocenter, Molecules. 19(2)
(2014) 1544-1567.
[16] B. Zhou, B. Zhang, X. Li, X. Liu, H. Li, D. Li, Z. Cui, H. Geng, L. Zhou, New 2-aryl-9-
methyl-β-carbolinium salts as potential acetylcholinesterase inhibitor agents: Synthesis,
bioactivity and structure–activity relationship, Sci. Rep. 8(1) (2018) 1559.
[17] Y. Rook, K-U. Schmidtke, F. Gaube, D. Schepmann, B. Wünsch, J. Heilmann, J.
Lehmannn, T. Winckler, Bivalent β-carbolines as potential multitarget anti-Alzheimer agents, J.
Med. Chem. 53(9) (2010) 3611–3617.
[18] Y. Schott, D. Michael, H. Rommelspacher, J. Lehmann, 6-Hydroxy- and 6-methoxy-β-
carbolines as acetyl- and butyrylcholinesterase inhibitors. Bioorg. Med. Chem. Lett. 16 (2006)
5840–5843.
[19] S. Ghosal, S.K. Bhattacharya, R. Mehta, Naturally occurring and synthetic β-carbolines as
cholinesterase inhibitors. J. Pharm. Sci. 61 (1972) 808–810.
[20] M. Zhang, D. Sun, Recent advances of natural and synthetic β-carbolines as anticancer
agents, Anticancer Agents Med. Chem. 15(5) (2015) 537-47.
[21] S. Kumar, A. Singh, K. Kumar, V. Kumar, Recent insights into synthetic β-carbolines with
anti-cancer activities, Eur. J. Med. Chem. 142 (2017) 48-73.
[22] T.B. Goh, R.Y. Koh, M.F. Yam, M.E. Azhar, M.N. Mordi, S.M. Mansor, 5-
Methoxytryptamine reacts with natural food flavour to produce 6-methoxy tetrahydro-β-
carbolines: In vitro investigation of their antioxidant and cytotoxicity properties, Food Chem.
183 (2015) 208-216.
[23] A. Spindler, K. Stefan, M. Wiese, Synthesis and investigation of tetrahydro-β-carboline
derivatives as inhibitors of the breast cancer resistance protein (ABCG2), J. Med. Chem. 59
(2016) 6121−6135.
[24] Bruker, SADABS, APEX2 and SAINT, Bruker AXS Inc, Madison, Wisconsin, USA, 2009.
[25] G.M. Sheldrick, A short history of SHELX, Acta Crystallogr. Sect. A 64 (2008) 112–122.
[26] G.M. Sheldrick, Crystal structure refinement with SHELXL, Acta Crystallogr. Sect. C 71
(2015) 3–8.
[27] A.L. Spek, Structure validation in chemical crystallography, Acta Crystallogr. Sect. D Biol.
Crystallogr. 65 (2009) 148–155.
[28] K.Y. Khaw, S.B. Choi, S.C. Tan, H.A. Wahab, K.L. Chan, V. Murugaiyah, Prenylated
xanthones from mangosteen as promising cholinesterase inhibitors and their molecular docking
studies, Phytomedicine 21 (2014) 1303–1309.
25

[29] G. Kryger, I. Silman, J.L. Sussman, Structure of acetylcholinesterase complexed with
E2020 (Aricept): Implications for the design of new anti-Alzheimer drugs, Structure 7(3) (1999)
297–307.
[30] P.V.R Shannon, W.M Leyshon, The structure and synthesis of a tetrahydro-β-carboline
alkaloid from Phalarisarundinacea: Some new tetrahydro-β-carbolines, J. Chem. Soc. C. 0 (1971)
2837-2839.
[31] P. Ashok, S. Chander, T.K. Smith, R.P. Singh, P.N. Jha, M. Sankaranarayanan, Biological
evaluation and structure activity relationship of 9-methyl-1-phenyl-9H-pyrido[3,4-b]indole
derivatives as anti-leishmanial agents. Bioorg. Chem. 84 (2019) 98-105.
[32] M. Bandini, A. Gualandi, M. Monari, A. Romaniello, D. Savoia, M. Tragni, Allylic
alcohols: Valuable synthetic equivalents of non-activated alkenes in gold-catalyzed
enantioselective alkylation of indoles, J. Organomet. Chem. 696(1) (2011) 338-347.
[33] B.V.S. Reddy, K. Kota, B.M. Rao, B. Sridhar, K. Mukkanti, Four-component, five-centered,
one-pot synthesis of 1-(1H-tetrazol-5-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole derivatives,
Tetrahedron Lett. 57(41) (2016) 4529–4532.
[34] M. Toganoh, N. Harada, H. Furuta, Hydrogen-bonding network in new scorpionate-type
ligand composed of pyridine/pyrrole hybrid and anion-binding behavior of the corresponding
rhodium complexes in alkyne cyclotrimerization reaction, J. Organomet. Chem. 693(19) (2008)
3141–3150.
[35] D. Cremer, J.A. Pople, General definition of ring puckering coordinates, J. Am. Chem. Soc.
97(6) (1975) 1354–1358.
[36] J. López-García, M. Lehocký, P. Humpolíček, P. Sáha, HaCaT keratinocytes response on
antimicrobial atelocollagen substrates: Extent of cytotoxicity, cell viability and proliferation, J.
Funct. Biomater. 5 (2014) 43-57.
[37] H. Guan, H. Chen, W. Peng, Y. Ma, R. Cao, X. Liu, A. Xu, Design of β-carboline
derivatives as DNA-targeting antitumor agents, Eur. J. Med. Chem. 41 (2006) 1167–1179.
[38] H. Gu, N. Li, J. Dai, Y. Xi, S. Wang, J. Wang, Synthesis and in vitro antitumor activity of
novel bivalent β-carboline-3-carboxylic acid derivatives with DNA as a potential target, Int. J.
Mol. Sci. 19(10) (2018) 3179.
[39] S.M. Orbach, R.R. Less, A. Kothari, P. Rajagopalan, In vitro intestinal and liver models for
toxicity testing, ACS Biomater. Sci. Eng. 3 (2017) 1898 −1910.
[40] G.F. Ecker, C.R. Noe, In silico prediction models for blood-brain barrier permeation, Curr.
Med. Chem. 11(12) (2004) 1617– 1628.
26

[41] C.P. Tang, Y. Ye, Y. Feng, R.J. Quinn, TCM, Brain function and drug space, Nat. Prod.
Rep. 33 (2016) 6–25.
[42] N.J. Abbott, Prediction of blood-brain barrier permeation in drug discovery from in vivo, in
vitro and in silico models. Drug Discov. Today Technol. 1(4) (2004) 407-416.
[43] M.S. Alavijeh, M. Chishty, M.Z. Qaiser, A.M. Palmer, Drug metabolism and
pharmacokinetics, the blood -brain barrier, and central nervous system drug discovery. NeuroRx.
2 (2005) 554–571.
[44] H. van de Waterbeemd, G. Camenisch, G. Folkers, J.R. Chretien, O.A. Raevsky, Estimation
of blood-brain barrier crossing of drugs using molecular size and shape, and H-bonding
descriptors, J. Drug Target. 6(2) (1998) 151-165.
[45] D.F. Veber, S.R. Johnson, H.Y. Cheng, B.R. Smith, K.W. Ward, K.D. Kopple, Molecular
properties that influence the oral bioavailability of drug candidates, J. Med. Chem. 45 (12)
(2002) 2615–2623.
[46] C. Lipinski, F. Lombardo, B. Dominy, P. Feeney, Experimental and computational
approaches to estimate solubility and permeability in drug discovery and development settings,
Adv. Drug Deliv. Rev. 23 (1997) 3–25.
[47] T. Osterberg, U. Norinder. Prediction of polar surface area and drug transport processes
using simple parameters and PLS statistics, J. Chem. Inf. Comput. Sci. 40 (2000) 1408–1411.
[48] P.D. Leeson, A.M. Davis. Time-related differences in the physical property profiles of oral
drugs, J. Med. Chem. 47 (2004) 6338–6348.
[49] H. Pajouhesh, G.R. Lenz, Medicinal chemistry properties of successful central nervous
system drugs, NeuroRx. 2 (2005) 541–553.
[50] L. Ioakimidis, L. Thoukydidiss, A. Mirzaa, S. Naeem, J. Reynisson, Benchmarking the
reliability of QikProp. Correlation between experimental and predicted values, QSAR Comb.
Sci. 27(4) (2008) 445-456.
27

Synthesis, characterization, crystal structure, in vitro
acetylcholinesterase inhibition and molecular docking studies of new
tetrahydro-β-carboline
Highlights:
•
Synthesis and spectroscopic analysis (IR, UV, 1D and 2D NMR) of a new tetrahydro-β-
carboline derivative (3).
•
•
•
•
X-ray crystallographic analysis of compound 3.
The AChE inhibition and cytotoxic studies of compound 3 and its intermediates.
Molecular docking of compound 3 on active site of AChE.
In silico prediction of BBB permeation and ADMET properties of title compound.