Neuritogenic activity of bi-functional bis-tryptoline triazole

Article abstract of DOI:10.1016/j.bmc.2016.12.027

Alzheimer's disease (AD) is a common neurodegenerative disorder, one of the hallmarks of which is the deposition of aggregated β-amyloid peptides (Aβ40,42) as plaques in the brain. Oligomers of these peptides have been reported to be toxic and to inhibit neurite outgrowth, as evidenced by neurite dystrophy and significant loss of synaptic connectivity of neurons in the AD brain resulting in cognitive decline. These peptides also react with biological metal in the brain to generate free radicals, thereby aggravating neuronal cell injury and death. Herein, multifunctional triazole-based compounds acting on multiple targets, namely β-secretase (BACE1), β-amyloid peptides (Aβ) as well as those possessing metal chelation and antioxidant properties, were developed and evaluated for neuritogenic activity in P19-derived neurons. At the non-cytotoxic concentration (1?nM), all multifunctional compounds significantly enhanced neurite outgrowth. New bis-tryptoline triazole (BTT) increased the neurite length and neurite number, by 93.25% and 136.09% over the control, respectively. This finding demonstrates the ability of multifunctional compounds targeting Aβ to enhance neurite outgrowth in addition to their neuroprotective action.

Full text of DOI:10.1016/j.bmc.2016.12.027

Accepted Manuscript
Neuritogenic Activity of Bi-Functional Bis-Tryptoline Triazole
Jutamas Jiaranaikulwanitch, Sarin Tadtong, Piyarat Govitrapong, Valery V.
Fokin, Opa Vajragupta
PII:
S0968-0896(16)30943-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.12.027
BMC 13454
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 October 2016
16 December 2016
20 December 2016
Please cite this article as: Jiaranaikulwanitch, J., Tadtong, S., Govitrapong, P., Fokin, V.V., Vajragupta, O.,
Neuritogenic Activity of Bi-Functional Bis-Tryptoline Triazole, Bioorganic & Medicinal Chemistry (2016), doi:
http://dx.doi.org/10.1016/j.bmc.2016.12.027
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Neuritogenic Activity of Bi-Functional Bis-
Tryptoline Triazole
Leave this area blank for abstract info.
Jutamas Jiaranaikulwanitcha,b,*, Sarin Tadtongc, Piyarat Govitrapongd, Valery V. Fokine and Opa Vajraguptab
HO
N
BTT
H
N
(S)
NH
NH
N
N
neuron
(S)
N
[BTT]
neurite
Bi-functional compound
BACE1 (IC50 = 16.73 μM)
Aβ aggregation (IC50 = 66.36 μM)
Neurite outgrowth activity

Bioorganic & Medicinal Chemistry
Neuritogenic Activity of Bi-Functional Bis-Tryptoline Triazole
Jutamas Jiaranaikulwanitcha,b,*, Sarin Tadtongc, Piyarat Govitrapongd, Valery V. Fokine and Opa
Vajraguptab
aDepartment of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand
bCenter of Excellent for Innovation in Drug Design and Discovery and Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, 447
Sri-Ayudhya Road, Bangkok 10400, Thailand
cDepartment of Pharmacognosy, Faculty of Pharmacy, Srinakharinwirot University, 63 Moo 7 Rangsit-Nakhonnayok Road, Ongkharak, Nakhonnayok 26120,
Thailand
dCenter for Neuroscience, Faculty of Science, Mahidol University, 272 Rama VI Road, Rajathevi, Bangkok 10400, Thailand
eDepartment of Chemistry, The Scripps Research Institute, 10500 North Torrey Pines Road, La Jolla, CA 92037, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Alzheimer’s disease (AD) is a common neurodegenerative disorder, one of the hallmarks of which
is the deposition of aggregated β-amyloid peptides (Aβ40,42) as plaques in the brain. Oligomers of
these peptides have been reported to be toxic and to inhibit neurite outgrowth, as evidenced by
neurite dystrophy and significant loss of synaptic connectivity of neurons in the AD brain resulting
in cognitive decline. These peptides also react with biological metal in the brain to generate free
radicals, thereby aggravating neuronal cell injury and death. Herein, multifunctional triazole-based
compounds acting on multiple targets, namely β-secretase (BACE1), β-amyloid peptides (Aβ) as
well as those possessing metal chelation and antioxidant properties, were developed and evaluated
for neuritogenic activity in P19-derived neurons. At the non-cytotoxic concentration (1 nM), all
multifunctional compounds significantly enhanced neurite outgrowth. New bis-tryptoline triazole
(BTT) increased the neurite length and neurite number, by 93.25% and 136.09% over the control,
respectively. This finding demonstrates the ability of multifunctional compounds targeting Aβ to
enhance neurite outgrowth in addition to their neuroprotective action.
Available online
Keywords:
Bis-tryptoline triazole
Multifunctional activity
Neuritogenic activity
β-secretase inhibitor
β-amyloid aggregation
2009 Elsevier Ltd. All rights reserved
rapid expression of Dickkopf-1 (Dkk1) in the Wnt signalling
pathway and Dkk1 causes synapse disassembly and synapse
1. Introduction
Alzheimer’s disease (AD) is the most common
neurodegenerative disorder characterised by synapse and neuron
loss with the accumulation of senile plaques and neurofibrillary
tangles.1 The aetiology of this disease has not been fully
understood; several aetiological factors have been implicated in
the pathogenesis, such as β-amyloid peptide (Aβ40, 42)
accumulation, iron deregulation, oxidative damage and
decreasing acetylcholine levels.2-4 The most interesting target
when searching for a new drug for AD treatment was β-amyloid.
It was recently found that not only amyloid plaques but also Aβ
peptides, as both soluble oligomers and insoluble fibrils, are toxic
and the causes of neuronal cell death.5 The effect of Aβ on
reducing neurite outgrowth has been reported in SH-SY5Y
human neuroblastoma cell lines6, mouse neuroblastoma cells7 and
chick sympathetic neurons.8 Moreover, Aβ oligomers induce the
loss.9 Hence, Aβ in oligomeric forms is more neurotoxic on
neurite outgrowth than high-molecular-weight aggregates or
fibril forms.10 Therefore, the inhibition of amyloid production to
reduce Aβ-mediated neuronal damage and death is the main
target of drug development for AD treatment, resulting in a large
number of inhibitors of β-secretase (BACE1) and β-amyloid
aggregation.11,12 Furthermore, other factors that aggravate Aβ-
induced neurotoxicity, such as metals10 and free radicals13, are the
emerging targets of multifunctional drug development for AD.
Metals accelerate Aβ-aggregation and produce reactive species or
free radicals when interacting with Aβ.14,15
In our previous research, the multifunctional compounds of
tryptoline and tryptamine triazole derivatives have been
discovered to be multifunctional neuroprotective, acting on
 Corresponding author. Tel.: +6653-944382-; fax: +6653-944380; e-mail: jutamas.jia@cmu.ac.th.

multiple targets, namely β-secretase (BACE1), Aβ, metal and
reactive species. Interestingly, there are three compounds (6h,
12c and 12h) which not only showed a neuroprotective effect
against Aβ-induced neuronal cell death but also increased cell
viability more than that of the control.14 Thus, it would be
interesting to explore whether these multifunctional compounds
(6h, 12c and 12h) are able to enhance neurite outgrowth. In
addition, the novel bis-tryptoline triazole (BTT) was designed
focusing on inhibitory action against BACE1 and Aβ aggregation
because of the poor inhibitory action of compounds 6h against
BACE1 and 12c against Aβ aggregation. The newly synthesised
bis-tryptoline triazole, BTT was evaluated for the multifunctional
modes of action neuritogenic activity together with the
previously reported compounds 6h, 12c and 12h (Figure 1).
population size, 5,000,000 per run of energy evaluations and
27,000 of the maximum number of generations.
2.3 Synthesis
Compound BTT was synthesised by Cu(I)-catalysed azide
alkyne cycloaddition reaction between the azide 5 and alkyne 3i
(Figure 2). Compound 5 was prepared in four steps as previously
reported.15 First, tryptoline-3-carboxylic acid was reacted with
methanol using acid catalysis to yield compound 2. Second, the
methyl ester of compound 2 was converted to alcohol by sodium
borohydride to obtain compound 3. Third, the amino group of
compound 3 was protected and the hydroxyl group was
converted to azide; finally, the amino group of compound 4 was
deprotected yielding compound 5. Alkyne 3i was prepared by the
reaction between compound 3 and propargyl bromide (Figure 2).
2.3.1 (S)-(2-(Prop-2-ynyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indol -3-yl) methanol (3i)
Compound 3 (0.3526 g, 1.74 mmol), sodium bicarbonate
(0.1754 g, 2.01 mmol) and propargyl bromide (0.22 mL, 2.02
mmol) were stirred in 10 mL DMF at 70°C for 3 hours. Then, 30
mL water was added to the reaction and the reaction was
extracted with ethyl acetate (20 mL x 3). The combined organic
phase was washed with brine, dried over sodium sulphate and
evaporated under reduced pressure. The obtained residue was
purified by column chromatography on silica gel
(EtOAc/CHCl3/NH4OH 2:8:0.1) to yield yellow oil (0.1415 g,
34%); Rf = 0.33 (CH2Cl2/EtOAc/NH4OH 3:7:0.1); FTIR (ATR)
(cm-1): 3545 (O-H, st), 3394 (indole N-H, st), 3282 (alkyne C-H,
st), 3051 (aromatic C-H, st), 2922, 2845 (aliphatic C-H, st), 2103
(C≡C, st), 1660 (C-N, bending), 1451 (aromatic C=C, st), 1327
(O-H, bending), 1223 (C-O, st), 1155 (C-N, st), 1068 (C-O, st),
Figure 1. Structures of multifunctional compounds BTT, 6h, 12c and 12h
2. Experimental
2.1 General
Molecular dockings were performed to determine the binding
mode of BTT to BACE1 and Aβ by the AutoDock program suite
version 4.2 on the Garibaldi platform at The Scripps Research
Institute. BTT was generated and optimised with ChemDraw
Ultra 9.0 and Chem3D Ultra 9.0. Chemical reagents were
purchased form Aldrich, Chem-Impex or AK Science. 1H-NMR
and 13C-NMR spectra were acquired on Bruker Avance 300 or
400 MHz instruments. Mass spectra were recorded on either a
Thermo Finnigan or LCMS Bruker MicroTof. IR spectra were
recorded on Nicolet FTIR 550. BACE1 enzyme and BACE1
substrate were purchased from Sino Biological® and
Calbiochem®, respectively. Amyloid-β (1–42) from Anaspec®
was used in ThT. Statistics were determined by t-test calculated
by OriginPro 8.5.1 and SPSS 17.0.
1
742 (aromatic C-H, bending); H-NMR (400 MHz, CDCl3) 7.82
(s, 1H, H9): δ, 7.44 (d, J = 7.63 Hz, 1H, H5), 7.30 (d, J = 7.95
Hz, 1H, H8), 7.15 (t, J = 7.47, 7.47 Hz, 1H, H7), 7.09 (t, J =
7.22, 7.22 Hz, 1H, H6), 4.08 (d, J = 16.61 Hz, 1H, H1b), 4.01 (d,
J = 16.36 Hz, 1H, H1a), 3.71 (dd, J = 6.57, 1.86 Hz, 2H, H10),
3.64 (dd, J = 16.70, 2.39 Hz, 1H, H11a), 3.43 (dd, J = 16.69, 2.33
Hz, 1H, H11b), 3.38-3.32 (m, 1H, H3), 2.86 (dd, J = 16.07, 5.47
Hz, 1H, H4b), 2.59 (dd, J = 16.08, 6.01 Hz, 1H, H4a), 2.26 (t, J =
2.33, 2.33 Hz, 1H, H13), 2.25-2.03 (br, 1H, OH); LRMS (ESI)
m/z 241.25 [M+H].
2.2 In Silico Studies
2.3.2 (S)-3-((4-((S)-(3-Hydroxymethyl-2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indol-2-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)-
2, 3,4,9-tetrahydro-1H-pyrido[3,4-b]indole, (BTT)
2.2.1 Docking Study of β-Secretase
BACE1 template (2IRZ-F), template was constructed from
two crystal structures of BACE1 bound to inhibitors (Protein
Data Bank code: 2IRZ16 and 1FKN17 as previously described.15
Docking parameters in the in silico studies were 100 run of
genetic algorithm (GA), 150 of population size, 15,000,000 per
run of energy evaluations and 27,000 of the maximum number of
generations.
A mixture of compound 5 (0.0983 g, 0.43 mmol), compound
3i (0.1250 g, 0.52 mmol), 5% mol CuSO4 and 20% mol sodium
ascorbate in 10 mL of t-BuOH/H2O/EtOH (2:2:1) was stirred at
room temperature for 24 hours. After ethanol was evaporated out,
water (10 mL) was added to the reaction. The aqueous solution
was extracted with ethyl acetate (10 mL x 3). The organic phase
was dried over sodium sulphate, concentrated and purified by
column chromatography (EtOAc/MeOH/NH4OH; 9.5:0.5:0.1). A
light brown powder of BTT was obtained (0.0746 g, 37%); Rf =
0.27 (EtOAc/MeOH/NH4OH; 9:1:0.1); m.p. 178-180 ºC; FTIR
(KBr) (cm-1): 3398 (O-H, s), 3056 (aromatic C-H, st), 2917, 2844
(aliphatic C-H, st), 1630 (N-H, bending), 1451 (aromatic C=C,
2.2.2 Docking Study of Amyloid β
The Aβ template (1Z0Q-1) was constructed from crystal
structures of the Aβ monomer (Protein Data Bank code: 1Z0Q18)
as previously described.14 Docking parameters in the in silico
studies were 100 run of genetic algorithm (GA), 150 of

st), 1330 (O-H, bending), 1229 (C-O, st), 1150 (C-N, st), 1049
(C-O, st), 748 (aromatic C-H, bending); H-NMR (400 MHz,
1H, H3″), 2.78 (dd, J = 15.6, 4.8 Hz, 1H, H4″b), 2.67 (dd, J =
15.2, 4.0 Hz, 2H, H4″a, H4b), 2.52-2.46 (m, 1H, H4a); 13C-NMR
(400 MHz, DMSO-d6): δ 145.148, 136.454, 136.362, 132.282,
131.519, 127.561, 127.103, 125.135, 121.169, 120.811, 118.92,
118.660, 117.776, 117.707, 111.445, 111.316, 105.954, 105.573,
61.215, 59.125, 53.779, 53.253, 47.838, 46.045, 41.995, 24.889,
21.113; LRMS (ESI) m/z 468.50 [M+H]; HRMS (ESI) m/z
calcd for [M+] 467.5655, Found 468.2499 [M+H].
1
DMSO-d6): δ 10.81 (s, 1H, H9), 10.68 (s, 1H, H9″), 8.13 (s, 1H,
H5′), 7.36 (d, J = 8.00 Hz, 1H, H5), 7.32 (d, J = 7.60 Hz, 1H,
H5″), 7.26 (t, J = 7.60, 7.60 Hz, 2H, H8, H8″), 7.03-6.96 (m, 2H,
H7, H7″), 6.43-6.90 (m, 2H, H6, H6″), 4.70 (br, 1H, OH), 4.60
(d, J = 6 Hz, 2H, H10), 4.02 (s, 2H, H1), 3.97 (d, J = 14 Hz, 1H,
H6′a), 3.91 (d, J = 14 Hz, 1H, H6′b), 3.78 (d, J = 5.6 Hz, 2H,
H1″), 3.72-3.51 (m, 2H, H10″), 3.50 (m, 1H, H3), 3.17-3.11 (m,
Figure 2. Synthesis of compound BTT. Reagents and conditions: (a) H2SO4, methanol, reflux, 18 h; (b) NaBH4, ethanol, THF, reflux, 18 h; (c) 4-
nitrobenzenesulfonyl chloride, TEA, CH2Cl2, rt, 4 h; (d) NaN3, DMF, 70°C, 6 h; (e) K2CO3, thiophenol, DMF, 50°C, 2 h; (f) alkyne 3i, CuSO4·5H2O, sodium
ascorbate, DMSO, rt, 24 h; (g) propargyl bromide, NaHCO3, DMF, 70°C, 3 h.
µL of 0.2 mM ferrous sulphate solution. Then, DI water was
added to adjust the volume to 150 μL. The mixtures were
2.4 Biological Activity Assay
incubated at room temperature for 10 min. After incubation, 50
μL of 1 mM ferrozine was added to the reaction mixture. The
2.4.1 β-Secretase Inhibition Assay
The novel compound BTT was evaluated for BACE1
inhibitory action at a concentration of 25 μM. Briefly, the
reaction wells containing 30 µL BACE1 enzyme stock solution
absorbance was measured at 562 nm by using infinite 200 pro™,
Tecan.14
0.01 unit/μL, 20 μL of 5% DMSO of test compound and 50 µL of
50 μM β-secretase substrate IV from Calbiochem® were
incubated at 37ºC for 30 min. The fluorescence was measured at
2.4.4 Free Radical Scavenging Assay
The compound BTT was prepared in 50% DMSO at 500 μM
and evaluated for antioxidant activity at 100 µM. The reaction
well containing 70 μL of DPPH solution (500 μM in methanol),
10 μL of methanol and 20 μL of test compound was incubated at
room temperature in dark for 30 min. The absorbance was
measured at 517 nm by using infinite 200 pro™, Tecan.14
Ex = 380 nm and Em = 510 nm by using SpectraMax Gemini
EM™.14 Then, β-secretase inhibitor IV was used as positive
control. The assays were run in triplicate. The IC50 values were
calculated by using GraphPad Prism 4 in nonlinear regression
curve fit.
2.4.5 Neuritogenic Activity Assay
2.4.2 Amyloid β Aggregation Inhibition Assay
P19 cells were grown in alpha minimal essential medium (α-
MEM) supplemented with 7.5% newborn calf serum (NCS),
2.5% foetal bovine serum (FBS), and 1% antibiotics-antimycotic
solution in a 5% CO2 humidified atmosphere, at 37ºC. Cells in
monolayer cultures were maintained in exponential growth by
subculturing every 2 days.19 Exponentially grown cultures were
trypsinised and dissociated into single cells. P19 cells (2 x 106
cells/mL) were then suspended in 10 mL α-MEM supplemented
with 5% FBS, 1% antibiotics-antimycotic solution and 0.5 µM all
The novel compound BTT was evaluated for the Aβ anti-
aggregation at 100 μM. In each well of a transparent plate, 9 µL
of 25 μM working solution of amyloid-β (1-42) peptide
(Anaspec®) and 1 µL of the test compound were mixed gently
and incubated in the dark at 37°C for 48 h. After incubation, 200
μL of 5 μM ThT (Sigma®) in 50 mM Tris buffer (pH 7.4) was
added to each well. The fluorescence was measured at Ex = 446
nm and Em = 490 nm by using SpectraMax Gemini EM™.14
trans-retinoic acid (RA) and seeded onto
a
100-mm
2.4.3 Fe (II) Chelation Assay
bacteriological culture dish. The cells formed large aggregates in
suspension. After 4 days of RA treatment, aggregates were
dissociated by 5-mL glass measuring pipette, re-plated on poly-
The test compound was prepared in 50% DMSO at 100 μM.
This solution (40 μL) was added to reaction wells containing 50

L-lysine-pre-coated multi-well plates (multi-well plates were
coated with 50 µg/mL poly-L-lysine dissolved in PBS for
overnight and sterilised under UV light for 30 min) at 7 x 104
cells/mL (150 µL/well in 96-well plate and 1.5 mL/well in 6-well
plate), in α-MEM supplemented with 10% FBS and 1%
antibiotics-antimycotic solution, and incubated for 24 h.
Cytosine-1-β-D-arabinoside or Ara-C (10 µM) was added at day
one after plating and the medium was changed every 2-3 days.
The differentiated neuronal cells, P19 derived-neurons, were used
after day 14 of the differentiation process.19-21
a phase-contrast microscope was observed. The appearance of
P19-derived neurons was compared to the control and measured
for the length and number of neurites. The average length and
number of neurites of 30 neurons from the assay were measured.
The data were expressed as the mean ± SEM (n = 1).22
2.4.6 Cell Viability Assay
The SH-SY5Y cells were cultured in minimum essential
medium (MEM): F-12 (1:1), 10% fetal bovine serum, MEM non-
essential amino acids (0.5×), 0.5 mM sodium pyruvate and 100
units/ml of penicillin and 100 μg/mL of streptomycin. The cells
were incubated under a humidified with 5% CO2 at 37 °C.
Viability assay: The assay was carried out on P19-derived
neurons cultured in
a 96-well plate. After 14 days of
SH-SY5Y cells were seeded in 96-well plates (2 × 104 cells
per well) for 24 h. Then, cells were treated with 10 μM of test
compounds for 2 h prior to exposure to 1 μM aggregated β-
amyloid (1-42). The preparation of aggregated β-amyloid was
done in a medium without serum, penicillin and streptomycin at
37 °C for 72 h. The aggregated β-amyloid treated cells were
incubated for 24 h. After 24 h, 15 μL of MTT reagent (5 mg/mL
MTT in serum free medium containing 10 μM HEPES) was
added to each well and incubated at 37°C for 3 h. The medium
was removed and 100 μL of 0.04 N HCl in isopropanol was
added. The absorbance was measured at Ex = 570 nm and Em =
630 nm using a Synergy™ HT multi-detection.23
differentiation, the α-MEM supplemented with 10% FBS, 10 µM
Ara-C and 1% antibiotics-antimycotic solution was removed and
DMSO solutions of the sample, diluted with α-MEM
supplemented with 10% FBS and 1% antibiotics-antimycotic
solution in the presence of 10 µM Ara-C, were added to give the
concentrations of 0.0001, 0.001, 0.01, 0.1, 1, and 10 µM. The
concentration of DMSO was added to the cultures at 0.5% v/v.
The α-MEM supplemented with 10% FBS, 10 µM Ara-C, and
1% antibiotics-antimycotic solution was added into control wells.
The cells were incubated for 18 h at 37ºC. Then, 150 µL of the
medium was removed, and 50 µL of XTT solution (1 mg/mL
XTT in 60ºC α-MEM + 25 µM PMS) was added. After
incubation at 37ºC for 4 hours, the 100 µL of PBS (phosphate
buffer saline solution) pH 7.4 was added. The OD value was
determined on a microplate reader at 450 nm. The data were
expressed as the mean ± SEM (n = 3, each n was run in
triplicate), with the medium as a control representing 100% cell
viability. The concentration that enhanced survival of cultured
neurons more than control will be further investigated for
neuritogenicity.22
Results and Discussion
Bis-Tryptoline Triazole (BTT) was designed to increase
BACE1 inhibitory action by replacing the phenolic motif of
compound 6h with the tryptoline, a core structure of previously
reported BACE1 inhibitors as displayed in Figure 3.15 This
modification altered the orientation of the structure and increased
the hydrophobicity with intended to improve binding affinity.
Moreover, the length between terminal aromatic rings was 13.2
Å, within the 13-14 Å, the optimal length for anti-Aβ aggregation
activity.14
Neuritogenicity assay: The assay was carried out with P19-
derived neurons cultured in a 6-well plate. The assay was
performed in a replicate. After 14 days of differentiation, the α-
MEM supplemented with 10% FBS, 10 µM Ara-C, and 1%
antibiotics-antimycotic solution was removed and DMSO
solution of the sample, diluted with the α-MEM supplemented
with 10% FBS, 10 µM Ara-C, and 1% antibiotics-antimycotic
solution, were added to give a final concentration of the samples
(6h, 12c, 12h and BTT) at concentrations that enhanced the
survival of cultured neurons more than the control. The
concentration of DMSO was added to the cultures at 0.5% v/v.
The α-MEM supplemented with 10% FBS, 10 µM Ara-C, and
1% antibiotics-antimycotic solution was added to control wells.
The cells were incubated for 18 h at 37ºC. The morphology under
BTT was synthesised and its multifunctional mode of activity
was determined in comparison with the previously reported
compounds 6h, 12c and 12h. The results in Figure 4 show that
BTT possessed multifunctional activity with different degrees of
potency between different actions i.e. high to moderate potency
against BACE1 and amyloid aggregation, moderate to weak for
metal chelation and antioxidant activity. As expected, the activity
of BTT against BACE1 was higher than 6h (IC50 16.73 µM vs. >
50 M) and the %inhibition of Aβ aggregation of BTT was
greater than that of compound 12c.
Figure 3. The structural design of BTT

Figure 4. The multifunctional activity of BTT and related compounds; BACE1 inhibition was evaluated at 25 µM while other activities were tested at 100 µM
To gain further insight, the binding modes of all
multifunctional compounds to the targets, BACE1 and Awere
determined by molecular docking. All compounds are located in
the substrate binding site of BACE1; BTT and 12c occupy three
pockets: S1, S2 and S4, while only two pockets are occupied by
compounds 6h (S1 and S2) and 12h (S1 and S3) (Figure 5A).
BTT interacted with Asp228, the catalytic amino acid, and
Gly230 via hydrogen bonds. The increase in BACE1 inhibitory
action of BTT compared to that of compounds 6h and 12h
possibly results from the insertion of tryptoline moieties into S2
and S4 pockets (Tyr71, Thr72, Gln73, Gly230, Thr231, Thr232,
Asn233 and Ser325), which provides the hydrophobic
interactions and consequently leads to the increase in binding
affinity (Figure 5B). The IC50 value of BTT (IC50 = 16.73 μM) is
comparable with that of 12c (IC50 = 20.75 µM) according to the
similar binding mode.
Figure 5. The binding modes of BTT (yellow) to two targets showing interacting amino acid and H-bond (dotted line): (A-B) BACE1 and (C) Aβ-amyloid
superimposed with compounds 6h (cyan), 12c (magenta) and 12h (green).

All multifunctional compounds showed significant increase in
neurite number, 113-136% increase when compared with control.
Moreover, the number of neurites was significantly greater in
cells treated with multifunctional compounds than that of
quercetin and geldanamycin treated control. The neurons
incubated with test compounds increased the neurite length up to
57-93% compared with control (Table 1).
For A aggregation, the modification did not improve the
anti-amyloid aggregation of BTT because the binding mode of
BTT with Aβ was similar to other multifunctional compounds.
BTT formed four hydrogen bonds with Glu11, His14, Glu22, and
Asp23 and interacted with Gln15, Val18, Phe19, Phe20, Glu22,
and Asp23, which are the key amino acids in the self-aggregation
region (Figure 5C). The docking results of all compounds against
BACE1 and amyloid beta aggregation are shown in Table S1.
Only compounds BTT and 12c, which showed good inhibition
against BACE1, significantly increased neurite length more than
quercetin and geldanamycin, the well-reconized neuroprotectors
and neurite outgrowth inducers. Taken together, compound BTT
showed the best neuritogenic activity by increasing both neurite
length and number. Between multifunctional modes of activity, it
appeared that BACE1 inhibitory activity played a significant role
in neurite outgrowth, as BTT with anti-amyloid aggregation (IC50
= 66.36 μM) moderate showed greater neuritogenic activity than
quercetin, a potent amyloid aggregation inhibitor (IC50 = 7.94
μM).30
These multifunctional compounds (BTT, 6h, 12c and 12h)
were evaluated for the effect on neurite outgrowth of P19-derived
neurons using quercetin and geldanamycin as positive controls.
The P19 cell line was selected in this experiment because P19
cells can differentiate to cholinergic neurons under 4-5 days of
retinoic acid treatment.20 Thus, this P19 cell line was suitable as
an activity testing model for AD. Quercetin was selected as a
reference compound because its effects are related to our
designed compounds namely antioxidant, amyloid aggregation
inhibitor and neurite outgrowth inducer.24-26 Quercetin mediated
its neurite outgrowth inducing effect through the elevation of
intracellular cAMP and GP-43 expression levels.26 Geldanamycin
(GA) was also included as a positive control as it possessed
neuroprotection against neuronal plaque formation in animal
models27,28 as well as augmented neurite outgrowth in vitro and
accelerate axonal regeneration in vivo.29 The exact mechanism of
GA is still not fully revealed, the plausible mechanisms of GA is
binding to heat shock protein 90 (Hsp90)27 and upregulation of
heat shock protein 72.28,29 Before the evaluation of BTT for
neuritogenic activity, the viability assay was performed at six
serial concentrations (0.0001, 0.001, 0.01, 0.1, 1, and 10 µM).
The results are shown in supplementary data. All compounds
were non-toxic at 1 nM and significantly increased the neuronal
viability at p-value < 0.05 (Figure 6).
The neuritogenic activity was determined at a lowest non-
cytotoxic concentration (1 nM); the morphology, length and
numbers of neurites were observed under a phase-contrast
microscope after treatment with the test compounds (Figure S3).
Figure 6. The effect of compounds (1 nM) on the neuronal viability of P-19
derived neurons. The data were expressed as the mean ± SEM (n = 3, each n
was run in triplicate), with the medium as a control representing 100% cell
viability using t-test analysis with *p < 0.05.
Table 1. The neuritogenic activity at concentration of 1 nM in P19-derived neurons
Neurite length
Neurite number
Average of  SE
Compounds
%
Average  SE (m)
%
Increase
Increase
Control
0.5% DMSO
BTT
0.00
1.89
0.00
5.65
68.70  6.37
70.00  5.50
2.30  0.21
2.43  0.23
132.76  13.12*,**
108.23  9.44*,**
126.50  10.08*,**
111.44  8.86*,**
109.33  9.06*,**
108.80 ± 19.86 *,**
5.43  0.26*,**
5.13  0.38*,**
5.03  0.25*,**
5.33  0.39*,**
3.57  0.34*,**
3.23 ± 0.28*,**
93.25
57.54
84.13
62.21
59.14
58.37
136.09
123.04
118.70
131.74
55.22
40.43
6h
12c
12h
Geldanamycin
Quercetin
*p < 0.05 compared with control, **p < 0.05 compared with 0.5% DMSO
t-Test was calculated by OriginPro 8.5.1

In addition, the increasing length of neuron cells is related to
BACE1 inhibitory activity: BTT > 12c > 12h > 6h. This result
might involve an increase of soluble β-amyloid precursor protein
alpha (sAPPα) by the inhibition of BACE1, as reported by the
research group of Mattsson and Porteius.31 This sAPPα can
stimulate neurite outgrowth by binding with p75NTR.32 Moreover,
contactin-2 has been recently reported to be the substrate of
BACE1. The role of contactin-2 is regulating axon guidance,
neurite outgrowth and cell adhesion.33
nanomolar level. The results reported herein support the validity
of the multifunctionality approach in drug development for AD.
Acknowledgements
The authors would like to acknowledge financial support
from the Office of the High Education Commission and Mahidol
University under the National Research Universities and the
Commission on Higher Education (grant number CHE-RG-2551-
53).
Supplementary data
Apart from BACE1, the neuritogenic activity might
contribute to anti-amyloid aggregation action, as compounds 6h
and 12h with moderate inhibition effects on BACE1 were able to
enhance the neurite outgrowth. Amyloid aggregation inhibitory
activity might partially contribute to the neuritogenic activity.
This inhibition of amyloid formation might reduce the activation
of Rho GTPase activity yielding decreasing of Rho Kinase
(ROCK II), the collapsin response mediator protein-2 (CRMP-2)
phosphorylate enzyme. The phosphorylation of CRMP-2 reduced
tubulin assembly in neurites causing microtubule disassembly.34,
References
1.
2.
Selkoe, D. J. Physiol. Rev. 2011, 81, 741-766.
Jakob-Roetne, R.; Jacobsen, H. Angew. Chem. Int. Ed. 2009, 48,
3030-3059.
3.
4.
5.
6.
Mandel, S.; Amit, T.; Bar-Am, O.; Youdim, M.B.H. Prog.
Neurobiol. 2007, 82, 348-360.
Cavalli, A.; Bolognesi, M.L.; Minarini, A.; Rosini, M.; Tumiatti, V.;
Recanatini, M.; Melchiorre, C. J. Med. Chem. 2008, 51, 347-372.
Ono, K.; Condron, M. M.; Teplow, D. B. Proc. Natl. Acad. Sci. USA.
2009, 106,14745-14750.
Lambert, M. P.; Stevens, G.; Sabo, S.; Barber, K.; Wang, G.; Wade,
W.; Krafft, G.; Snyder, S.; Holzman, T. F.; Klein, W. L. J. Neurosci.
Res. 1994, 39, 377-385.
35
It is plausible that the inhibitions of BACE1 and amyloid
aggregation are the key factors in the promotion of neuron
outgrowth. In addition, BTT was determined for the protective
effect against amyloid-induced toxicity in SH-SY5Y neuronal
cells using curcumin, a potent antioxidant and anti-amyloid
aggregation (IC50 = 0.63 μM)36 as control. The result showed that
BTT was able to protect neurons against amyloid-induced
toxicity and increased cell viability over control significantly
(Figure 7).
7.
8.
9.
Eraser, P. E.; Lévesque, L.; McLachlan, D. R. J. Neurochem. 1994,
62, 1227-1230.
Postuma, R. B.; He, W.; Nunan, J.; Beyreuther, K.; Masters, C. L.;
Barrow, C. J.; Small, D. H. J. Neurochem. 2000, 74,1122-1130.
Purro, S. A.; Dickins, E. M.; Salinas, P. C. J. Neurosci. 2012, 32,
3492 –3498.
10. Tabner, B. J.; Mayes, J.; Allsop, D. Int. J. Alzheimers. Dis. 2011,
546380.
11. Ghosh, A. K.; Osswald, H. L. Chem. Soc. Rev. 2014, 43(19), 6765-
6813.
12. Nie, Q.; Du, X.,G.; Geng, M. Y. Acta. Pharmacol. Sin. 2011, 32,
545–551.
13. Budimir, A. Acta. Pharm. 2011, 61, 1-14.
14. Jiaranaikulwanitch, J.; Govitrapong, P.; Fokin, V. V.; Vajragupta, O.
Molecules. 2012, 17, 8312-8333.
15. Jiaranaikulwanitch, J.; Boonyarat, C.; Fokin, V. V.; Vajragupta, O.
Bioorg. Med. Chem. Lett. 2010, 20, 6572-6576.
16. Rajapakse, H. A.; Nantermet, P. G.; Selnick, H. G.; Munshi, S.;
McGaughey, G. B.; Lindsley, S. R.; Young, M. B.; Lai, M. T.;
Espeseth, A. S.; Shi X. P.; Colussi, D.; Pietrak, B.; Crouthamel, M.
C.; Tugusheva, K.; Huang, Q.; Xu, M.; Simon, A. J.; Kuo, L.;
Hazuda, D. J.; Graham, S.; Vacca, J. P. J. Med. Chem. 2006, 49,
7270-7273.
Figure 7. The protective effect of BTT against Aβ induced neuronal toxicity
by MTT viability assay using t-test analysis with *p < 0.05, **p< 0.01 vs Aβ
treated cells. t-Test was calculated by SPSS 17.0.
17. Hong, L.; Koelsch, G.; Lin, X.; Wu, S.; Terzyan, S.; Ghosh, A. K.;
Zhang, X. C.; Tang, J. Science. 2000, 290,150-153.
18. Tomaselli, S.; Esposito, V.; Vangone, P.; van Nuland, N. A. J.;
Bonvin, A. M. J. J. ; Guerrini, R.; Tancredi, T.; Temussi, P. A.;
Picone, D. ChemBioChem. 2006, 7, 257-267.
19. Jones-Villeneuve, E. M.; McBurney, M. W.; Rogers, K. A.; Kalnins,
V. I. J. Cell. Biol. 1982, 94, 253-262.
20. MacPherson, P. A.; McBurney, M. W. Methods. 1995, 7, 238-252.
21. Jones-Villeneuve, E. M.; Rudnicki, M. A.; Harris, J. F.; McBurney,
M. W. Mol. Cell. Biol. 1983, 3, 2271-2279.
22. Tadtong, S.; Meksuriyen, D.; Tanasupawat, S.; Isobe, M.;
Suwanborirux, K. Bioorg. Med. Chem. Lett. 2007, 17, 2939-2943.
23. Mosmann, T. J .Immunol. Methods. 1983, 65, 55-63.
24. Matsuzaki, K.; Noguch, T.; Wakabayashi, M.; Ikeda, K.; Okada, T;
Ohashi, Y.; Hoshino, M.; Naiki, H. Biochim. Biophys. Acta. 2007,
1768(1), 122-130.
Beside amyloid generation and aggregation partway,
neuritogenicity of BTT might be form other mechanism because
BTT can increse cell viability in amyloid-induced toxicity over
the control significantly. Thus, other proposed neurite outgrowth
mechanism of BTT such as inducing of cAMP level and Gap-43
expression and decreasing of protein level (raf1, ERK, Akt1 and
CDK4) will be invatigated in further study. 26, 37
3. Conclusions
We successfully designed multifunctional compounds BTT,
6h, 12c and 12h to exert the neuritogenic activity at the

25. Tangsaengvit, N.; Kitphati, W.; Tadtong. S.; Bunyapraphatsara, N.;
Nukoolkarn, V. Evid. Based. Complement. Alternat. Med. 2013,
2013, 838051.
26. Chen, M. M.; Yin, Z. Q.; Zhang, L.Y.; Liao, H. Chin J Nat Med.
2015, 13(9), 667-672.
27. Sittler, A.; Lurz, R.; Lueder, G.; Priller, J.; Lehrach, H.; Hayer-Hartl,
M. K.; Hartl, F. U.; Wanker, E. E. Hum Mol Genet, 2001, 10(12),
1307-1315.
28. Herbst, M.; Wanker, E. E. Neurodegener Dis, 2007, 4(2-3), 254-260.
29. Sun, H. H.; Saheb-Al-Zamani, M.; Yan, Y.; Hunter, D. A.;
Mackinnon, S. E.; Johnson, P. J. Neuroscience. 2012, 223, 114-123.
30. Stefani, M.; Rigacci, S. Int. J. Mol. Sci. 2013, 14, 12411-12457.
31. Mattsson, N.; Rajendran, L.; Zetterberg, H.; Gustavsson, M.;
Andreasson, U.; Olsson, M.; Brinkmalm, G.; Lundkvist, J.;
Jacobson, L. H.; Perrot, L.; Neumann, U.; Borghys, H.; Mercken,
M.; Dhuyvetter, D.; Jeppsson, F.; Blennow, K.; Portelius, E. PLoS
One. 2012, 7, e31084.
32. Hasebe, N.; Fujita, Y.; Ueno, M.; Yoshimura, K.; Fujino, Y.;
Yamashita, T. PLoS One. 2013, 8, e82321.
33. Gautam, V.; D’Avanzo, C.; Hebisch, M.; Kovacs, D. M.; Kim, D. Y.
Mol. Neurodegener. 2014, 9, 4.
34. Petratos, S.; Li, Q. X.; George, A. J.; Hou, X.; Kerr, M. L.; Unabia,
S. E.; Hatzinisiriou, I.; Maksel, D.; Aguilar, M. I.; Small, D. H.
Brain. 2008, 131, 90-108.
35. Mokhtar, S. H.; Bakhuraysah, M. M.; Cram, D. S.; Petratos, S. Int. J.
Alzheimers. Dis. 2013, 910502.
36. Ono, K.; Haseqawa, K.; Naiki, H.; Yamada, M. J. Neurosci. Res.
2004, 75, 742-750.
37. Jin, E.; Sano, M. Cell Biochem Funct. 2008, 26(8), 825-832.
.