Discovery of neurotrophic agents based on hydroxycinnamic acid scaffold

Article abstract of DOI:10.1111/cbdd.12829

The number of people affected by neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease is rapidly increasing owing to the global increase in life expectancy. Small molecules with neurotrophic effects have great potential for management of these neurological disorders. In this study, different (C1–C12) alkyl ester derivatives of hydroxycinnamic acids (HCAs) were synthesized (a total of 30 compounds). The neurotrophic capacity of the test compounds was examined by measuring promotion of survival in serum-deprived conditions and enhancement of nerve growth factor (NGF)-induced neurite outgrowth in PC12 neuronal cells. p-Coumaric, ferulic, and sinapic acids and their esters did not alter cell survival, while caffeic acid and all its alkyl esters, especially decyl and dodecyl caffeate, significantly promoted neuronal survival at 25?μm. Methyl, ethyl, propyl, and butyl caffeate esters also significantly enhanced NGF-induced neurite outgrowth, among which the most effective ones were propyl and butyl esters, which at 5?μm led to 25- and 22-fold increases in the number of neurites, respectively. The findings of the docking study suggested phosphatidylinositol 3-kinase (PI3K) as the potential molecular target. In conclusion, our findings demonstrate that alkyl esters of caffeic acid can be useful as scaffolds for the discovery of therapeutic agents for neurodegenerative diseases.

Full text of DOI:10.1111/cbdd.12829

Received Date : 03-Apr-2016
Revised Date : 13-Jun-2016
Accepted Date : 11-Jul-2016
Article type : Research Article
Discovery of neurotrophic agents based on hydroxycinnamic acid scaffold
Razieh Hosseini1,2, Fatemeh Moosavi1,2, Hamid Rajaian2, Tiago Silva3, Diogo Magalhães e
Silva3, Pedro Soares3, Luciano Saso4, Najmeh Edraki1, Ramin Miri1, Fernanda Borges3*,
Omidreza Firuzi1*
1 Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
2
Department of Pharmacology, School of Veterinary Medicine, Shiraz University, Shiraz,
Iran
3
CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of
Porto, 4169-007 Porto, Portugal
4 Department of Physiology and Pharmacology "Vittorio Erspamer", Sapienza University of
Rome, Italy
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12829
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* Corresponding authors:
Omidreza Firuzi, MD PhD
Medicinal and Natural Products Chemistry Research Center
Shiraz University of Medical Sciences
Shiraz, Iran
Email: firuzio@sums.ac.ir
Fernanda Borges, PhD
CIQUP/Department of Chemistry and Biochemistry
Faculty of Sciences, University of Porto
4169-007 Porto, Portugal
E-mail: fborges@fc.up.pt
Abstract:
The number of people affected by neurodegenerative disorders such as Alzheimer’s disease
and Parkinson’s disease is rapidly increasing owing to the global increase in life expectancy.
Small molecules with neurotrophic effects have great potential for management of these
neurological disorders. In this study, different (C1-C12) alkyl ester derivatives of
hydroxycinnamic acids (HCAs) were synthesized (a total of 30 compounds). The
neurotrophic capacity of the test compounds was examined by measuring promotion of
survival in serum-deprived conditions and enhancement of nerve growth factor (NGF)-
induced neurite outgrowth in PC12 neuronal cells. p-Coumaric, ferulic and sinapic acids and
their esters did not alter cell survival, while caffeic acid and all its alkyl esters, especially
decyl and dodecyl caffeate, significantly promoted neuronal survival at 25 µM. Methyl, ethyl,
propyl and butyl caffeate esters also significantly enhanced NGF-induced neurite outgrowth,
among which the most effective ones were propyl and butyl esters, which at 5 µM led to 25-
and 22-fold increases in the number of neurites, respectively. The findings of the docking
study suggested phosphatidylinositol 3-kinase (PI3K) as the potential molecular target. In
conclusion, our findings demonstrate that alkyl esters of caffeic acid could be potentially
useful for discovery of therapeutic agents for neurodegenerative diseases.
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Keywords:
Neurodegenerative diseases, hydroxycinnamic acids, alkyl ester, neurotrophic, synthesis
Introduction
Neurodegenerative diseases are most common in the elderly and with the increase in life
expectancy in recent decades, these diseases have begun to impose a huge health burden
worldwide(1, 2). No effective disease-modifying treatment is yet available for the most
common neurodegenerative disorders, including Alzheimer’s disease and Parkinson’s
disease, therefore, there is a pressing and unmet need for new therapies that stop or reverse
the progression of these pathologies. Many investigations have targeted the accumulation of
toxic proteins in the central nervous system, including amyloid-β(3-5) and tau(5) in
Alzheimer’s disease and α-synuclein in Parkinson’s disease(6). However, these strategies
have failed to deliver an effective disease-modifying treatment so far(5, 7, 8).
Neurotrophic factors of the nervous system, such as nerve growth factor (NGF) and brain
derived neurotrophic factor (BDNF), play important roles in neuronal differentiation and
survival and are critical for the development and maintenance of the nervous system(9, 10).
The binding of neurotrophic factors to their receptors (tropomyosin receptor kinase, Trk
receptors) triggers signaling pathways that promote neuronal differentiation and survival(11,
12). Preclinical and clinical findings suggest that neurotrophins could be a promising therapy
for neurodegenerative diseases like Alzheimer’s disease(11, 13) and Parkinson’s disease(11,
14). However, neurotrophin peptides lack a suitable pharmacokinetic profile and hardly cross
the blood-brain barrier; therefore, efforts have been devoted to the search for small molecule
neurotrophic agents that mimic the action of neurotrophins or potentiate their effect(15-17).
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Several investigators have become increasingly interested in polyphenols over the past
decades, because of the abundance of these compounds in diet and their possible role in the
prevention of several pathologies such as cardiovascular diseases, cancer and
neurodegeneration(18). In particular, in the context of neurodegenerative diseases, several
polyphenols have been reported to induce neuronal differentiation and neurite outgrowth(19-
23). Hydroxycinnamic acids (HCAs) represent an important class of polyphenolic
compounds(24), which are widely distributed in plants and abundantly found in fruits and
vegetables and possess several biological activities(25-27). Several studies have shown that
phenolic compounds may exert their neurotrophic effect by the activation of neurotrophic
signalings such as Ras/MAPK/ERK or phosphatidylinositol 3-kinase (PI3K)/Akt
pathways(23). A number of studies have shown that PI3K and its downstream effector Akt
play an important role in mediating the neuronal survival and increased neurite outgrowth, as
well as other neurotrophic effects of polyphenols(28-31).
In continuation of our interest in the synthesis and evaluation of the biological activities of
HCAs(32-35), we have synthesized 30 HCA derivatives consisting of short and long alkyl
esters of p-coumaric acid, ferulic acid, sinapic acid and caffeic acid, and have tested their
ability to promote cell survival and induction of differentiation in neuronal cells. We further
performed a molecular docking simulation study in order to find out the binding potential of
active derivatives with PI3K as a putative molecular target.
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Materials and methods
Reagents
Dimethyl sulfoxide, methanol and propanol were obtained from Merck (Darmstadt,
Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) as well as
all reagents needed for chemical synthesis were obtained from Sigma-Aldrich (Lisbon, PT).
All reagents and solvents were pro analysis and were used without further purification. Fetal
bovine serum (FBS) and horse serum were acquired from Invitrogen (San Diego, CA), while
penicillin/streptomycin, RPMI 1640, sterile phosphate-buffered saline (PBS), and trypsin
EDTA 0.05% were purchased from Biosera (Ringmer, UK). Deionized water was used in all
the experiments.
Chemistry
Apparatus
1H and 13C NMR were acquired at room temperature on a Brüker Biospin GmbH 400
spectrometer operating at 400 and 101 MHz, respectively. Chemical shifts were expressed in
δ (ppm) values relative to tetramethylsilane (TMS) as internal reference; coupling constants
(J) were given in Hz. Assignments were also made from DEPT (Distortionless Enhancement
by Polarization Transfer) (underlined values). Microwave-assisted synthesis was performed
in Biotage® Initiator Microwave Synthesizer.
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Synthesis of alkylcinnamates
Method A - Synthesis of short chain alkyl cinnamates
The cinnamic acid (0.5 g), the appropriate alcohol (15 mmol) and 2 drops of concentrated
sulphuric acid were added to a glass vial (2-5 mL) and sealed. The system was heated to 20ºC
above the boiling point of the alcohol for 5 min(36). After cooling to room temperature, the
crude product was purified by flash chromatography using silica gel as stationary phase and
dichloromethane or ethyl acetate as eluent. Compounds CAF1-4, FER 1,2 and 4, SIN1-4
have been synthesized as previously described and the structural data was in accordance with
the previously reported data(32, 35).
1
Trans-Methyl-3-(4-hydroxyphenyl)propenoate (PCC1). Yield: 74%. H NMR(DMSO-
d6): 3.70 (3H, s,CH3), 6.40 (1H, d, J=16.0 H(2)), 6.80 (2H, m, H(3’’), H(5’’)), 7.50 (3H, m,
H(2’), H(6’), H(3)), 10.05 (1H, s, COOH). 13C NMR(DMSO-d6):51.3 (CH3), 113.9 (C(3’’),
C(5’’)), 115.8 (C(2)), 125.1 (C(1’)), 130.0 (C(2’’), C(6’’)), 144.8 (C(3)), 159.9 (C(4’’)),
167.1 (CO). MS/EI m/z: 178 (M+•, 24), 147 (100).
Trans-Ethyl-3-(4-hydroxyphenyl)propenoate (PCC2). Yield: 63%.1H NMR(DMSO-d6):
1.25 (3H, t, J=7.1, CH3), 4.16 (2H, t, J=7.1 Hz, CH2), 6.39 (1H, d, J=15.9, H(2)), 6.81 (2H,
m, H(3’’), H(5’’)), 7.56 (3H, m, H(2’), H(6’), H(3)), 10.04 (1H, s, COOH). 13C
NMR(DMSO-d6):14.8 (CH3), 59.5 (CH2), 114.3 (C(3’’), C(5’’)), 115.8 (C(2)), 125.1 (C(1’)),
130.3 (C(2’’), C(6’’)), 144.6 (C(3)), 159.9 (C(4’’)), 166.7 (CO). MS/EI m/z: 192 (M+•, 76),
147 (100).
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Trans-Hexyl-3-(4-hydroxy-3-methoxyphenyl)propenoate (FER5). Yield: 80%. 1H
NMR(CDCl3): 0.87 (3H, t, J=8.0, H(6’)), 1.32 (6H, bs, H(3’)-H(5’)), 1.65 (2H, m, H(2’)),
3.82 (3H, s, OCH3), 4.16 (2H, t, J=8.0 H(1’)), 6.26 (1H, d, J=16.0 H(2)), 6.87 (1H, d, J=8.0
H(6’’)), 6.99 (2H, m, H(2’’), H(5’’)), 7.58 (1H, d, J=16.0, H(3)). 13C NMR(CDCl3):14.0
(C(6’)), 22.5 (C(5’)), 25.6 (C(4’)), 28.7 (C(3’)), 31.5 (C(2’)), 55.8 (OCH3), 64.6 (C(1’)),
109.6 (C(2’’)), 115.0 (C(5’’)), 115.3 (C(2)), 122.9 (C(6’’)), 126.9 (C(1’’)), 144.9 (C(3)),
147.1 (C(4’’)), 148.2 (C(3’’)), 167.6 (CO). MS/EI m/z: 278 (M+•, 93), 194 (100).
Method B- Synthesis of long chain alkyl cinnamates
Part B1-Synthesis of malonic acid half esters
Equimolar quantities of Meldrum’s acid and of the appropriate alcohols were refluxed in
toluene (5 mL) for 4 h. After cooling the reaction to room temperature a solution of saturated
NaHCO3 was added to the mixture. The compounds were extracted with diethyl ether; the
organic layer was washed with water and dried with Na2SO4. The solvents were evaporated
to yield the malonic acid half esters. The compounds were used in the next reaction without
further purification.
Monohexyl malonate.1H NMR(CDCl3): 0.89 (3H, t, J=8.0 H(6’)), 1.33 (6H, bs, H(3’)-
H(5’)), 1.65 (2H, m, H(2’)), 3.43 (2H, s, H(2)), 4.16 (2H, t, J=8.0 H(1’)).
1
Monooctyl malonate. H NMR(CDCl3): 0.89 (3H, t, J=8.0 H(8’)), 1.30 (10H, bs, H(3’)-
H(7’)), 1.65 (2H, m, H(2’)), 3.43 (2H, s, H(2)), 4.16 (2H, t, J=8.0 H(1’)).
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Monodecyl malonate.1H NMR(CDCl3): 0.88 (3H, t, J=8.0 H(10’)), 1.30 (14H, bs, H(3’)-
H(9’)), 1.65 (2H, m, H(2’)), 3.43 (2H, s, H(2)), 4.16 (2H, t, J=8.0 H(1’)).
Monododecyl malonate.1H NMR(CDCl3): 0.89 (3H, t, J=8.0 H(12’)), 1.31 (18H, bs, H(3’)-
H(11’)), 1.68 (2H, m, H(2’)), 3.45 (2H, s, H(2)), 4.19 (2H, t, J=8.0 H(1’)).
Monohexadecyl malonate.1H NMR(CDCl3): 0.89 (3H, t, J=8.0 H(16’)), 1.30 (26H, bs,
H(3’)-H(15’)), 1.67 (2H, m, H(2’)), 3.45 (2H, s, H(2)), 4.19 (2H, t, J=8.0 H(1’)).
Part B2-Synthesis of alkyl chain cinnamates via Knoevenagel condensation
Equimolar quantities of half ester of malonic acid (1-5) and the appropriate benzaldehyde
derivative were added to cyclohexane (3-5 mL), and then anhydrous pyridine (1 equiv.) and
aniline (1.6 equiv.) were added to the mixture, and the mixture was refluxed for 6-10 h. After
cooling, the mixture was added in an ice bath concentrated HCl. After pH neutralization, the
mixture was extracted with diethyl ether and the organic layer was died with Na2SO4. The
solvent was evaporated under vacuum obtaining the crude product. The compounds were
purified by flash chromatography using gradient elution (petroleum ether/ ethyl acetate).
Trans-Hexyl-3-(4-hydroxyphenyl)propenoate (PCC5).Yield 45%.1H NMR(CDCl3): 0.90
(3H, t, J=8.0 H(6’)), 1.35 (6H, bs, H(3’)-H(5’)), 1.70 (2H, m, H(2’)), 4.20 (2H, t, J=8.0
H(1’)), 6.30 (1H, d, J=16.0 H(2)), 6.87 (2H, d, J=8.0 H(3’’), H(5’’)), 7.41 (2H, d, J=8.0
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13
H(2’’), H(6’’)), 7.63 (1H, d, J=16.0 H(3)). CNMR(CDCl3):14.0 (C(6’)), 22.7 (C(5’)), 25.7
(C(4’)), 28.7 (C(3’)), 31.5 (C(2’)), 65.0 (C(1’)), 115.3 (C(2)), 116.0 (C(3’’), C(5’’)), 126.9
(C(1’’)), 130.0 (C(2’’), C(6’’)), 144.9 (C(3)), 158.2 (C(4’)), 168.2 (CO). MS/EI m/z: MS/EI
m/z: 248 (M+•, 35), 147 (100).
Trans-Octyl-3-(4-hydroxyphenyl)propenoate (PCC6). Yield 40%.1H NMR(CDCl3):0.92
(3H, t, J=8.0 H(8’)), 1.35 (10H, bs, H(3’)-H(7’)), 1.73 (2H, m, H(2’)), 4.25 (2H, t, J=8.0
H(1’)), 6.33 (1H, d, J=16.0 H(2)), 6,95 (2H, d, J=8.0 H(3’’), H(5’’)), 7.43 (2H, d, J=8.0
H(2’’), H(6’’)), 7.68 (1H, d, J=16.0 H(3)). 13C NMR(CDCl3):14.1 (C(8’)), 22.7 (C(7’)), 26.0
(C(6’)), 28.7 (C(5’)), 29.2 (C(4’)), 29.3 (C(3’)), 31.8 (C(2’)), 65.2 (C(1’)), 114.7 (C(2)),
116.1 (C(3’’), C(5’’)), 126.5 (C(1’’)), 130.2 (C(2’’), C(6’’)), 145.5 (C(3)), 158.8 (C(4)),
168.8 (CO). MS/EI m/z: 276 (M+•, 21), 147 (100).
1
Trans-Decyl-3-(4-hydroxyphenyl)propenoate (PCC7). Yield34%. H NMR(CDCl3):0.88
(3H, t, J=8.0 H(10’)), 1.32 (14H, bs, H(3’)-H(9’)), 1.70 (2H, m, H(2’)), 4.20 (2H, t, J=8.0
H(1’)), 6.30 (2H, d, J=16.0 H(2)), 6.86 (2H, d, J=8.0 H(3’’), H(5’’)), 7.42 (2H, d, J=8.0
H(2’’), H(6’’)), 7.63 (1H, d, J=16.0 H(3)). 13C NMR(CDCl3):14.1 (C(10’)), 22.7 (C(9’)),
26.0 (C(8’)), 28.8 (C(7’)), 29.3 (C(6’), C(5’)), 29.6 (C(4’), C(3’)), 31.9 (C(2’)), 64.9 (C(1’)),
115.5 (C(2)), 115.9 (C(3’’), C(5’’)), 127.1 (C(1’’)), 130.0 (C(2’’), C(6’’)), 144.6 (C(3)),
158.0 (C(4’’)), 168.0 (CO). MS/EI m/z: 304 (M+•, 34), 147 (100).
Trans-Dodecyl-3-(4-hydroxyphenyl)propenoate
(PCC8).
Yield
30%.
1H
NMR(CDCl3):0.90 (3H, t, J=8.0 H(12’)), 1.33 (18H, H(3’)-H(11’)), 1.72 (2H, m, H(2’)), 4.22
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(2H, t, J=8.0 H(1’)), 6.32 (2H, d, J=16.0 H(2)), 6.89 (2H, d, J=8.0 H(3’’), H(5’’)), 7.44 (2H,
d, J=8.0 H(2’’), H(6’’)), 7.65 (1H, d, J=16.0 H(3)). 13C NMR(CDCl3):15.0 (C(12’)), 23.6
(C(11’)), 26.9 (C(10’)), 29.6 (C(9’)), 30.2 (C(8’)), 30.3 (C(7’)), 30.4 (C(6’)), 30.5 (C(5’)),
30.6 (C(4’), C(3’)), 32.8 (C(2’)), 65.8 (C(1’)), 116.2 (C(2)), 116.9 (C(3’’), C(5’’)), 127.8
(C(1’’)), 130.9 (C(2’’), C(6’’)), 145.8 (C(3)), 159.2 (C(4’’)), 169.1 (CO). MS/EI m/z: 332
(M+•, 40), 147 (100).
1
Trans-Hexyl-3-(3,4-dihydroxyphenyl)propenoate (CAF5). Yield 58%. H NMR(DMSO-
d6): 0.87 (3H, t, J=8.0 H(6’)), 1.31 (6H, bs, H(3’)-H(5’)), 1.62 (2H, m, H(2’)), 4.10 (2H, t,
J=8.0 H(1’)), 6.26 (1H, d, J=16.0 H(2)), 6.76 (1H, d, J=8.0 H(5’’)), 7.02 (2H, m, H(2’’),
13
H(6’’)), 7.47 (1H, d, J=16.0 H(3)), 9.12 (1H, s, OH), 9.57 (1H, s, OH). C NMR(DMSO-
d6):14.4 (C(6’)), 22.5 (C(5’)), 25.6 (C(4’)), 28.7 (C(3’)), 31.4 (C(2’)), 64.2 (C(2’)), 114.5
(C(2’’)), 115.3 (C(2)), 116.2 (C(5’’)), 121.8 (C(6’’)), 126.0 (C(1’’)), 145.5 (C(3)), 146.0
(C(3’’)), 148.8 (C(4’’)), 167.1 (CO). MS/EI m/z: 264 (M+•, 80), 180 (100).
1
Trans-Octyl-3-(3,4-dihydroxyphenyl)propenoate (CAF6).Yield 53%. H NMR(DMSO-
d6): 0.86 (3H, t, J=8.0 H(8’)), 1.30 (10H, bs, H(3’)-H(7’)), 1.62 (2H, m, H(2’)), 4.10 (2H, t,
J=8.0 H(1’)), 6.26 (1H, d, J=16.0 H(2)), 6.76 (1H, d, J=8.0 H(5’’)), 7.02 (2H, m, H(2’’),
H(6’’)), 7.47 (1H, d, J=16.0 H(3)), 9.40 (2H, bs, 2xOH). 13C NMR(DMSO-d6): 15.3 (C(8’)),
23.5 (C(7’)), 26.8 (C(6’)), 29.7 (C(5’)), 30.0 (C(4’), C(3’)), 32.6 (C(2’)), 65.1 (C(1’)), 115.4
(C(2’’)), 116.2 (C(2)), 117.1 (C(5’’)), 122.7 (C(6’’)), 126.7 (C(1’’)), 146.4 (C(3)), 146.9
(C(3’’)), 149.7 (C(4’’)), 168.0 (CO). MS/EI m/z: 292 (M+•, 50), 180 (100).
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1
Trans-Decyl-3-(3,4-dihydroxyphenyl)propenoate (CAF7). Yield 42%. H NMR(CDCl3):
0.88 (3H, t, J=8.0 H(10’)), 1.32 (14H, bs, H(3’)-H(9’)), 1.70 (2H, m, H(2’)), 4.20 (2H, t,
J=8.0 H(1’)), 6.27 (1H, d, J=16.0 H(2)), 6.88 (1H, d, J=8.0 H(5’’)), 7.01 (1H, dd, J=8.0; 4.0
H(6’’)), 7.12 (1H, d, J=4.0 H(2’’)), 7.59 (1H, d, J=16.0 H(3)). 13C NMR(CDCl3): 14.1
(C(10’)), 22.7 (C(9’’)), 26.0 (C(8’’)), 28.7 (C(7’’)), 29.3 (C(6’’), C(5’’)), 29.6 (C(4’’),
C(3’’)), 31.9 (C(2’’)), 65.0 (C(1’’)), 114.5 (C(2’’)), 115.5 (C(5’’), 115.6 (C(2)), 122.4
(C(6’’)), 127.5 (C(1’’)), 143.9 (C(3’’)), 145.0 (C(3)), 146.4 (C(4’’)), 168.1 (CO). MS/EI
m/z:320 (M+•, 28), 180 (100).
Trans-Dodecyl-3-(3,4-dihydroxyphenyl)propenoate
(CAF8).
Yield
50%.
1H
NMR(DMSO- d6): 0.85 (3H, t, J=8.0 H(12’)), 1.24 (18H, bs, H(3’)-H(11’)), 1.61 (2H, m,
H(2’)), 4.10 (2H, t, J=8.0 H(1’)), 6.25 (1H, d, J=16.0 H(2)), 6.76 (1H, d, J=8.0 H(5’’)), 7.00
(2H, m, H(2’’), H(6’’)), 7.46 (1H, d, J=16.0 H(3)), 9.20 (1H, bs, OH), 9.60 (1H, bs, OH). 13C
NMR(DMSO-d6): 15.3 (C(12’)), 23.5 (C(11’)), 26.8 (C(10’)), 29.6 (C(9’)), 30.0 (C(8’)), 30.1
(C(7’)), 30.3 (C(6’), C(5’)), 30.4 (C(4’), C(3’)), 32.7 (C(2’)), 65.1 (C(1’)), 115.4 (C(2’’)),
116.2 (C(2)), 117.1 (C(5’’)), 122.7 (C(6’’)), 126.9 (C(1’’)), 146.4 (C(3)), 146.9 (C(3’’)),
149.7 (C(4’’)), 168.0 (CO). MS/EI m/z: 348 (M+•, 45), 180 (100).
Trans-Decyl-3-(4-hydroxy-3-methoxyphenyl)propenoate (FER7). Yield 34%. 1H
NMR(CDCl3): 0.90 (3H, t, J=8.0 H(10’)), 1.35 (14H, bs, H(3’)-H(9’)), 1.71 (2H, m, H(2’)),
3.93 (3H, s, OCH3), 4.21 (2H, t, J=8.0 H(1’)), 6.31 (1H, d, J=16.0 H(2)), 6.93 (1H, d, J=8.0
H(5’’)), 7.07 (2H, m, H(2’’), H(6’’)), 7.63 (1H, d, J=16.0 H(3)). 13C NMR(CDCl3):14.1
(C(10’)), 22.7 (C(9’)), 26.0 (C(8’)), 28.8 (C(7’)), 29.3 (C(6’), C(5’)), 29.6 (C(4’), C(3’)), 31.9
(C(2’)), 56.0(OCH3), 64.7 (C(1’)), 109.4 (C(2’’)), 114.8 (C(5’’)), 115.7 (C(2)), 123.0
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(C(6’’)), 127.1 (C(1’’)), 144.7 (C(3)), 146.8 (C(4’’)), 148.0 (C(3’’)), 167.4 (CO). MS/EI m/z:
334 (M+•, 79), 194 (100).
Trans-Dodecyl-3-(4-hydroxy-3-methoxyphenyl)propenoate (FER8). Yield 27%. 1H
NMR(CDCl3): 0.90 (3H, t, J=8.0 H(12’)), 1.33 (18H, bs, H(3’)-H(11’)), 1.72 (2H, m, H(2’)),
3.95 (3H, s, OCH3), 4.21 (2H, t, J=8.0 H(1’)), 6.31 (1H, d, J=16.0 H(2)), 6.94 (1H, d, J=8.0
H(5’’)), 7.07 (2H, m, H(2’’), H(6’’)), 7.63 (1H, d, J=16.0 H(3)). 13C NMR(CDCl3):14.3
(C(12’)), 22.9 (C(11’)), 26.2 (C(10’)), 29.0 (C(9’)), 29.5 (C(8’), C(7’)), 29.7 (C(6’), C(5’)),
29.8 (C(4’), C(3’)), 32.1 (C(2’)), 56.1 (OCH3), 64.8 (C(1’)), 109.5 (C(2’’)), 114.9 (C(5’’)),
115.9 (C(2)), 123.2 (C(6’’)), 127.2 (C(1’’)), 144.8 (C(3)), 146.9 (C(4’’)), 148.1 (C(3’’)),
167.6 (CO). MS/EI m/z: 362 (M+•, 99), 194 (100).
Trans-Octyl-3-(4-hydroxy-3,5-dimethoxyphenyl)propenoate (SIN6). Yield 48%. 1H
NMR(CDCl3): 0.91 (3H, t, J=8.0 H(8’)), 1.36 (10H, bs, H(3’)-H(7’)), 1.72 (2H, m, H(2’)),
3.94 (6H, s, 2xOCH3), 4.22 (2H, t, J=8.0 H(1’)), 6.33 (1H, d, J=16.0 H(2)), 6.80 (2H, s,
H(2’’), H(6’’)), 7.61 (1H, d, J=16.0 H(3)). 13C NMR(CDCl3):14.1 (C(8’)), 22.7 (C(7’)), 26.0
(C(6’)), 28.8 (C(5’)), 29.2 (C(4’), C(3’)), 31.8 (C(2’)), 56.4 (2xOCH3), 64.7 (C(1’)), 105.1
(C(2’’), C(6’’)), 116.1 (C(2’)), 126.2 (C(1’’)), 137.1 (C(4’’)), 144.8 (C(3)), 147.2 (C(3’’),
C(5’’)), 167.3 (CO). MS/EI m/z: 336(M+•, 100)
Trans-Decyl-3-(4-hydroxy-3,5-dimethoxyphenyl)propenoate
(SIN7).Yield
37%.1H
NMR(CDCl3): 0.88 (3H, t, J=8.0 H(10’)), 1.32 (14H, bs, H(3’)-H(9’)),1.70 (2H, m, H(2’)),
3.91(6H, s, 2xOCH3), 4.19 (2H, t, J=8.0 H(1’)), 6.31 (1H, d, J=16.0 H(2)), 6.77 (2H, s,
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H(2’’), H(6’’)), 7.59 (1H, d, J=16.0 H(3)). 13C NMR(CDCl3): 14.1 (C(10’), 23.0 (C(9’)),
26.0 (C(8’)), 28.8 (C(7’)), 29.3 (C(6’), C(5’)), 29.6 (C(4’), C(3’)), 31.9 (C(2’)), 56.3
(2xOCH3), 64.7 (C(1’)), 105.1 (C(2’’), C(6’’)), 116.1 (C(2)), 126.0 (C(1’’)), 137.1 (C(4’’)),
144.8 (C(3)), 147.2 (C(3’’), C(5’’)), 167.3 (CO). MS/EI m/z: 365 (M+•, 100).
1
Trans-Dodecyl-3-(4-hydroxy-3,5-dimethoxyphenyl)propenoate (SIN8). Yield 30%. H
NMR(CDCl3): 0.90 ((3H, t, J=8.0 H(12’)), 1.34 (18H, bs, H(3’)-H(11’)), 1.71 ((2H, m,
H(2’)), 3.94(6H, s, 2xOCH3), 4.21 (2H, t, J=8.0 H(1’)), 6.33 (1H, d, J=16.0 H(2)), 6.79 (2H,
s, H(2’’), H(6’’)), 7.61 (1H, d, J=16.0 H(3)). 13C NMR(CDCl3):15.0 (C(12’)), 23.6 (C(11’)),
26.9 (C(10’)), 29.7 (C(9’)), 30.2 (C(8’)), 30.3 (C(7’)), 30.5 (C(6’), C(5’)), 30.6 (C(4’), C(3’)),
32.8 (C(2’)), 57.2 (2xOCH3), 65.6 (C(1’)), 105.9 (C(2’’), C(6’’)), 117.0 (C(2)), 126.9
(C(1’’)), 138.0 (C(4’’)), 145.7 (C(3)), 148.1 (C(3’’), C(5’’)), 168.2 (CO). MS/EI m/z: 392
(M+•, 100).
Cell Culture
PC12 cells (rat pheochromocytoma) were a generous gift from Professor Lloyd A. Greene
(Department of Pathology and Cell Biology, Columbia University, New York, NY, USA).
Cells were grown in RPMI 1640 medium, supplemented with 10% heat-inactivated horse
serum, 5% heat-inactivated fetal bovine serum, 100 U/mL penicillin G, and 100 mg/mL
streptomycin. Cells were cultured on plates coated with collagen obtained from rat tail and
were grown at 37 ºC in humidified air containing 5% CO2. Two-thirds of the growing
medium was changed every 2–3 days and the cells were subcultured once a week.
Cell viability assay
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The effect of synthesized cinnamic acid derivatives on the viability of the PC12 cells was
determined by the MTT reduction assay, which is based on the reduction of a tetrazolium salt
by mitochondrial dehydrogenase in viable cells(37, 38). The cells were seeded in collagen
coated 96-well microplates at a density of 6 × 105 cells/mL (80 µL per well) in serum free
medium. Ten μL of NGF dissolved in RPMI at 5 or 50 ng/ml or the test compounds at the
final concentrations of 1, 5 and 25 µM were added in triplicate. Test compound were first
dissolved in DMSO and then diluted in growth medium at least 1000 times. After incubation
of cells at 37 ◦C for 48 h, the media were removed from the wells and 20 μL of MTT solution
(0.5 mg/mL) was added to each well and incubated for another 90 min. DMSO (200 μL/well)
was added to each well and plates were further incubated at 37 ◦C for 60 min and then placed
on a shaker for another 30 min. The absorbance was measured with a microplate reader
(Model 680, Bio-Rad) at 570 nm with background correction at 650 nm.
Quantification of neurite outgrowth in PC12 cells
PC12 cells were seeded in collagen-coated 12-well plates in RPMI medium supplemented
with 2% horse serum and 1% fetal bovine serum at a density of 5×104 cells/mL (1 mL per
well). After 24 h of incubation, different concentrations of the test compounds (1, 5 and 25
µM) with or without NGF (50 and 5 ng/mL) were added to the wells. After 3 days of
incubation, the growth medium was refreshed with the same concentrations of the test
compounds and NGF. The numbers of neurites were counted in at least five randomly
selected microscopic fields by visual examination after 8 days. Neuronal processes with a
length equal to or longer than a cell body were counted as neurites. The total numbers of
neurites as well as those equal or longer than 100 µm were counted separately. Microscopic
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observations were made with an inverted Nikon microscope (model eclipse TS100-F) using
phase contrast objectives. Each experiment was repeated at least 3 times.
Molecular docking study
Molecular docking study was performed using Autodock 4.2 and Auto Dock Tools 1.5.4
(ADT). For this purpose, the X-ray crystal structure of PI3K carrying wortmannin as cognate
ligand in binding site (PDB code: 1E7U, downloaded from protein data bank,
http://www.rcsb.org) was used for molecular docking study and investigation of biding
interactions of CAF analogues with PI3K active site as their possible target was performed.
The PDB file of protein was regenerated by removing water molecules and cognate ligand,
adding polar hydrogens and computing Collman charges. The 3D structures of CAF and
CAF1-8 were created using Chem3D Ultra 12.0 software. Each ligand was separately
submitted to a molecular mechanics conformational search (MM+ force field) and the
geometry of the most stable conformer was further optimized by semi-empirical method
(AM1) in Hyperchem software. The Gasteiger charges were added and the degrees of
torsions were defined to generate PDBQT format of the ligands. The grid parameter file was
generated using AutoGrid. Numbers of points in x, y, z dimensions were set to 60*60*60
with 0.375 A ° grid spacing. Rigid macromolecule was chosen to define the docking
parameter file using Lamarckian Genetic Algorithm (LGA), with 100 GA runs and 25000000
energy evaluations while the other parameters were left as default.
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Statistical analysis
Values were expressed as mean S.E.M. from 3-4 independent determinations. Statistical
analyses were carried out by analysis of variance (ANOVA) followed by appropriate post hoc
tests including multiple comparison tests (LSD). All analyses were made using the SPSS
statistical software package (Version 11.5 for Windows). Differences with probability values
of less than 0.05 were considered as statistically significant.
Results
Chemistry
In order to evaluate the influence of esterification on the neurotrophic activity, a set of alkyl
cinnamate derivatives based on p-coumaric acid, ferulic acid, sinapic acid and caffeic acid
were synthesized (Figure 1). Short chain length alkyl esters were obtained following a
microwave-assisted Fischer esterification of the acid with the appropriate alcohol, as
previously described (Scheme 1A)(39). Long alkyl chain ester derivatives were prepared by a
Knoevenagel-Doebner condensation between the benzaldehyde with the appropriate aromatic
pattern and the previously prepared malonate half ester with the desired tether length
(Scheme 1B)(40). The compounds were obtained in moderate to high yields and fully
13
characterized by magnetic resonance spectroscopy (1HRMN, CRMN and DEPT135) and
mass spectrometry.
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Hydroxycinnamic acids and their alkyl esters promote survival in serum deprived PC12
neuronal cells
Survival of serum-deprived PC12 cells exposed to synthesized compounds was evaluated
using (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (MTT) assay. As
expected, NGF at the concentrations of 5 and 50 ng/mL significantly promoted PC12 cells
survival as compared to untreated serum-deprived control cells (Figure 2). On the other hand,
non-esterified p-coumaric acid, ferulic acid and sinapic acid at the concentrations of 1, 5 and
25 μM did not have any effect on the survival. Similarly, exposure to p-coumarate, ferulate,
and sinapate esters at the same concentrations did not enhance viability. However, caffeic
acid and its esters (CAF1, CAF2, CAF3, CAF4, CAF5, CAF6, CAF7, CAF8) at the
concentration of 25 μM, but not at 1 and 5 μM, significantly promoted the survival of PC12
cells. CAF7 and CAF8 were the most active compounds and could enhance the cell survival
up to more than 250% compared to the control. Interestingly, the effects of these last 2 esters
were stronger than the effect of nerve growth factor (NGF) at the concentrations of 5 and 50
ng/mL.
Caffeate ester derivatives enhance neurite outgrowth induced by NGF in PC12 cells
The effect of caffeate esters on the NGF-induced neurite outgrowth in PC12 cells was
examined. Treatment of cells with NGF alone (5 and 50 ng/mL) induced the outgrowth of
neurites in the cells (Figures 3 and Table 1). When PC12 cells were exposed to NGF 5 ng/mL
co-treated with methyl (CAF1) and ethyl caffeate (CAF2) at the concentrations of 5 and 25
μM, and also propyl (CAF3) and butyl caffeate (CAF4) at the concentration of 5 μM, the
total number of neurites significantly increased compared to the cells that were exposed to
NGF 5 ng/mL alone. Similarly, the number of longer neurites (longer than 100 µm)
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significantly increased in cells co-treated with methyl, ethyl, propyl and butyl caffetaes at 5
μM (Figures 3 and Table 1). The concomitant treatment of cells with NGF and the rest of the
caffeate esters including hexyl (CAF5), octyl (CAF6), decyl (CAF7) and doceyl (CAF8)
esters as well as caffeic acid did not significantly change the number of neurites compared to
NGF treatment alone (data not shown).
Molecular docking study of CAFs with PI3K
According to previous reports, PI3K might serve as a probable molecular target of
polyphenolic compounds underlying the neuroprotective potential of these structures(41, 42).
In order to gain insight on binding interactions of CAFs with PI3K active site, we performed
a molecular docking study using Autodock 4.2 software with flexible ligand/rigid
macromolecule procedure based on the crystal structure of PI3K (PDB ID: 1E7U) with its
cognate ligand, wortmannin. Docking optimization and validation was performed by
redocking of the co-crystallized conformation of cognate ligand into the PI3K binding site.
Consequently, RMSD (root mean square deviation) of the best-docked conformation of the
native ligand from the experimental one was used for validation of the docking study(43).
The best-docked and experimental conformation of wotmannin correlated quite well with an
RMSD of 1.73Ǻ and binding free energy of -8.96 Kcal/mol (Table 2).
Docking results of different CAF analogues are illustrated in Table 3. Based on the obtained
data, the introduction and elongation of alkyl side chains into caffeic acid structure resulted in
an enhanced binding interaction of CAF alkyl esters with PI3K active site (∆Gb= -5.05 to -
7.33 Kcal/mol). Moreover, phenolic hydroxyl substitutes were involved in key hydrogen
binding with Asp841 and Asp964 of the active site in all alkyl ester derivatives of caffeic acid
and elongation of the alkyl ester pendant indicated further binding interactions with Met953
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and Glu880 of catalytic site in most cases. The binding mode of CAF7 with PI3K binding
site is depicted in Figure 4. CAF7 was well accommodated in the active site and was
involved in key hydrogen bond interactions with Val882 Asp841, and Asp964 (respective
binding distances were 1.70, 1.33 and 1.35 Å) and hydrophobic interactions with residues of
the catalytic site including Phe961, Met953, Val882 and Trp812.
Discussion
In this study, 30 derivatives of HCAs including p-coumaric acid, ferulic acid, sinapic acid
and caffeic acid and their C1-C12 alkyl esters were synthesized and their neurotrophic
capacities were examined in PC12 neuronal cells. Caffeic acid esters were the most efficient
neurotrophic compounds.
We first screened the protective effects of the synthesized compounds against serum
deprivation in PC12 cells by MTT assay. Several studies have used serum deprivation and
MTT assay to test the neurotrophic effect of various compounds (44-46). Our findings
showed that caffeic acid and its esters significantly enhanced cell viability and inhibited cell
death induced by serum deprivation at the concentration of 25 µM. Decyl and dodecyl
caffeates exhibited the strongest neuroprotective activities, followed by shorter esters C1-C4.
On the other hand, p-coumaric acid, ferulic acid and sinapic acid ester derivatives did not
show any significant neuroprotective activity.
Furthermore, the influence of caffeate esters on the neurite outgrowth of PC12 cells was also
examined and it was observed that they intensely enhanced the effect of low dose NGF. The
most prominent effects belonged to CAF3 and CAF4 derivatives, which at 5 μM
considerably increased the total number of neurites (25- and 22-fold increases, respectively)
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and the number of neurites longer than 100 µm (36- and 34-fold increases, respectively)
compared to NGF alone. Although long alkyl chain caffeate esters (CAF7 and CAF8 esters)
were the most active agents in promotion of neuronal survival, they did not show any
significant effect on neuritogeneis. Other studies have similarly shown that phenolic
compounds such as green tea polyphenols(47), isorhamnetin, a flavonol(48), and genistein,
an isoflavone(49), potentiated the action of NGF and induced neuritogenesis in PC12 cells.
We had previously reported that short alkyl esters of caffeic acid significantly inhibited
hydrogen peroxide-induced neuronal PC12 cell death, but ferulate esters could not reduce
oxidative stress-induced cell damage(35). Lipophilicity is an important physicochemical
property of phenolic compounds and several lines of evidence have shown that esterification
of phenolic compounds can increase the lipophilicity and therefore their pharmacological
activity (33, 34, 50, 51).
The neuroprotective effect of caffeic acid phenetyl ester (CAPE) has been shown in rat
organotypic midbrain slice cultures and in an animal model of neurodegeneration induced by
LPS and 6-hydroxydopamine(52). Furthermore, a recent study has shown that CAPE induces
neurite outgrowth in PC12 cells and protects these cells from MPP(+) neurotoxin-induced
damage(53).
HCA derivatives have been reported to have diverse biological activities including anticancer
effect (54) by different mechanism including inhibition of 17β-hydroxysteroid
dehydrogenases (55). In this study, only caffeic acid ester derivatives containing catechol
moiety in their structures exhibited the biological effect and other HCAs did not show
significant activities. This structural feature may guide the discovery of selective agents.
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The neuroprotective properties of NGF and its potential implication in treatment of
neurodegenerative diseases are well established(56, 57); however, pharmacokinetic
limitations and side effects such as neuropathic pain have hindered the therapeutic application
of NGF peptide(58). Hence, small molecular weight neurotrophic agents that are devoid of
the limitations of endogenous neurotrophins could be of therapeutic value in
neurodegenerative diseases.
Several studies have shown that phenolic compounds may exert their neurotrophic effect by
the activation of neurotrophic signaling pathways such as PI3K/Akt and ERK1/2 (23).
PI3K/Akt and ERK1/2 signaling pathways can promote cell survival in the nervous system as
well as other tissues(59, 60). Various studies have shown that PI3K and its downstream
effector Akt, are involved in neuronal survival and increasing neurite outgrowth as well as
other neurotrophic effects of polyphenols(28-31).
Possible mechanisms that might take place at a molecular level underling the neurotrophic
effects of HCAs might be comparable to those associated with compounds with similar
structures: Caffeoylserotonin has shown protective effects against oxidative stress-induced
cell death in keratinocytes through the activation of PI3K/Akt pathway and subsequent
activation of Nrf2(61). In another study, caffeic acid ester fraction (a mixture of different
esters of caffeic acid mainly consisting of quinic acid esters) has shown protective effects in
primary rat astrocytes against hypoxia through the production and release of neurotrophins by
stimulation of CREB and Akt signaling pathways(62). Protective effects of caffeic acid and
CAPE against acrolein-induced neurotoxicity in mouse hippocampal neuronal cells has also
been shown to be mediated by MAPKs and Akt signaling(63).
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On the other hand, although ferulic acid and its derivatives did not show significant activities
in our neuronal cell model, other investigators applying different methodologies have
reported that ferulic acid exerts cytoprotective effects against oxidative stress in umbilical
vein endothelial cells by stimulation of PI3K and ERK pathways(42). Another study on
ferulic acid conjugated to tacrine, has also shown neuroprotective effects in attenuation of 6-
hydroxydopamine-induced apoptosis in PC12 cells through the stimulation of Akt
pathway(64). Furthermore, it has been reported that sodium ferulate exerts protective effects
against glutamate toxicity in cortical neurons by modulation of PI3K and the MEK/ERK
signaling pathways(65) and against amyloid-β-induced neurotoxicity by activation of Akt in
rat hippocampal neurons(66).
We performed a docking study to assess the interaction of caffeate esters with PI3K, as a
potential target. The findings of the docking study clearly showed that caffeate esters had a
reasonable interaction with the active site of PI3K and the binding energies of the ester
derivatives correlated well with the results of the survival promotion assay.
In conclusion, our findings demonstrate the neurotrophic effects of caffeic acid ester
derivatives, among which methyl, ethyl, propyl and butyl esters have the most prominent
capacities. The findings of the docking study also suggest that PI3K may be a molecular
target that mediates the neurotrophic action of these compounds. These derivatives could
form the basis for the rational design of neurotrophic agents with potential applications for
management of neurodegenerative diseases.
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Acknowledgements
The authors wish to thank the Iranian National Science Foundation (Grant number:
92024294) and the Natural Antimicrobials Centre of Excellence for the financial supports of
this project. We also thank the Foundation for Science and Technology (FCT) of Portugal
(PEst-C/QUI/UI0081/2015) and QREN (FCUP-CIQ-UP-NORTE-07-0124-FEDER- 000065)
for financial support and grant of T. Silva (SFRH/BD/79671/2011). This study was part of
the PhD thesis of Razieh Hosseini.
Conflict of interests
The authors declare that they have no conflicts of interest.
Figure captions:
Scheme 1. Synthetic strategies used for the synthesis of short chain (1A) and long chain alkyl
cinnamates (1B) (i) Meldrum’s acid, toluene, reflux, 4h; (ii) benzaldehyde derivative, aniline,
cyclohexane, reflux, 6-10h.
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Figure 1. Chemical structure of hydroxycinnamic acids and the alkyl ester derivatives
under study.
Figure 2: Hydroxycinnamic acids and their alkyl esters promote survival in serum
deprived PC12 neuronal cells.
PC12 cells were seeded in 96-well plates deprived of serum and were immediately treated
with NGF (5 or 50 ng/mL) or hydroxycinnamic acids and their alkyl esters [p-coumarate (A),
ferulate (B), sinapate (C) and caffeate esters (D)] at different concentrations. After 48 h, cell
viability was measured by the MTT assay. The results are plotted as the percentage of cell
survival compared to untreated cells and expressed as mean S.E.M of 3-4 independent
experiments. * Significantly different from untreated control cells (P<0.05).
Figure 3: Caffeic acid and its alkyl esters enhance NGF-induced neurite outgrowth in
PC12 cells.
PC12 cells were seeded in 12-well plates and treated with either NGF alone or in concomitant
incubation with caffeic acid and caffeate alkyl esters (CAF, CAF1-12) at the concentrations
of 1, 5 and 25 μM. The images were taken after 8 days of incubation. Representative images
are shown for the most active caffeate alkyl esters (CAF1, CAF2, CAF3 and CAF4).
Figure 4. Best docking pose of CAF7 into the active site of PI3K.
The ligand is depicted in stick mode and the interaction residues are demonstrated in line
style. Hydrogen bond interactions are depicted as green lines.
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References
1. Bonaconsa M, Colavito V, Pifferi F, Aujard F, Schenker E, Dix S, et al. (2013) Cell clocks
and neuronal networks: neuron ticking and synchronization in aging and aging-related
neurodegenerative disease. Curr Alzheimer Res;10: 597-608.
2. Olesen J, Gustavsson A, Svensson M, Wittchen HU, Jönsson B. (2012) The economic cost
of brain disorders in Europe. Eur J Neurol;19: 155-62.
3. Selkoe DJ. (2007) Developing preventive therapies for chronic diseases: lessons learned
from Alzheimer's disease. Nutr Rev;65: S239-S43.
4. Lukiw WJ. (2012) Amyloid beta (Aβ) peptide modulators and other current treatment
strategies for Alzheimer's disease (AD). Expert Opin Emerg Drugs;17: 43-60.
5. Hampel H, Schneider LS, Giacobini E, Kivipelto M, Sindi S, Dubois B, et al. (2014)
Advances in the therapy of Alzheimer's disease: targeting amyloid beta and tau and
perspectives for the future. Expert Rev Neurother;15: 83-105.
6. Grelle G, Otto A, Lorenz M, Frank RF, Wanker EE, Bieschke J. (2011) Black tea
theaflavins inhibit formation of toxic amyloid-β and α-synuclein fibrils. Biochemistry;50:
10624-36.
7. Löhle M, Reichmann H. (2010) Clinical neuroprotection in Parkinson's disease—still
waiting for the breakthrough. J Neurol Sci;289: 104-14.
8. Tofaris GK, Schapira AH. (2015) Neurodegenerative diseases in the era of targeted
therapeutics: how to handle a tangled issue. Mol Cell Neurosci;66: 1-2.
9. Bothwell M. (2014) NGF, BDNF, NT3, and NT4. NGF, BDNF, NT3, and NT4. Springer:
p. 3-15.
10. Ivanov A. (2014) The Role of NGF and BDNF in Mature Brain Activity Regulation. Zh
Vysshei Nerv Deiat Pavlova;64: 137-46.
11. Weissmiller AM, Wu C. (2012) Current advances in using neurotrophic factors to treat
neurodegenerative disorders. Transl Neurodegener;1: 14.
12. Reichardt LF. (2006) Neurotrophin-regulated signalling pathways. hilos Trans R Soc
Lond B Biol Sci;361: 1545-64.
13. Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM, et al.
(2009) Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate
models of Alzheimer's disease. Nat Med;15: 331-7.
14. Rangasamy SB, Soderstrom K, Bakay RA, Kordower JH. (2010) Neurotrophic factor
therapy for Parkinson’s disease. Prog Brain Res;184: 237-64.
15. Price RD, Milne SA, Sharkey J, Matsuoka N. (2007) Advances in small molecules
promoting neurotrophic function. Pharmacol Ther;115: 292-306.
16. Saragovi HU, Gehring K. (2000) Development of pharmacological agents for targeting
neurotrophins and their receptors. Trends Pharmacol Sci;21: 93-8.
17. Scarpi D, Cirelli D, Matrone C, Castronovo G, Rosini P, Occhiato E, et al. (2012) Low
molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity. Cell
Death Dis;3: e339.
18. Del Rio D, Rodriguez-Mateos A, Spencer JP, Tognolini M, Borges G, Crozier A. (2013)
Dietary (poly) phenolics in human health: structures, bioavailability, and evidence of
protective effects against chronic diseases. AntioxidnRedox Signal;18: 1818-92.
19. Lai H-C, Wu M-J, Chen P-Y, Sheu T-T, Chiu S-P, Lin M-H, et al. (2011) Neurotrophic
effect of citrus 5-hydroxy-3, 6, 7, 8, 3′, 4′-hexamethoxyflavone: promotion of neurite
outgrowth via cAMP/PKA/CREB pathway in PC12 cells. PLoS One;6: e28280.
This article is protected by copyright. All rights reserved.

20. Tangsaengvit N, Kitphati W, Tadtong S, Bunyapraphatsara N, Nukoolkarn V. (2013)
Neurite outgrowth and neuroprotective effects of quercetin from Caesalpinia mimosoides
Lamk. on cultured P19-derived neurons. Evid Based Complement Alternat Med;2013:
21. Lin L-F, Chiu S-P, Wu M-J, Chen P-Y, Yen J-H. (2012) Luteolin induces microRNA-132
expression and modulates neurite outgrowth in PC12 cells. PloS one;7: e43304.
22. Zhang Z, Cai L, Zhou X, Su C, Xiao F, Gao Q, et al. (2015) Methyl 3, 4-
dihydroxybenzoate promote rat cortical neurons survival and neurite outgrowth through the
adenosine A2a receptor/PI3K/Akt signaling pathway. NeuroReport;26: 367-73.
23. Moosavi F, Hosseini R, Saso L, Firuzi O Modulation of neurotrophic signaling pathways
by polyphenols. Drug Des Devel Ther;10: 23.
24. Manach C, Scalbert A, Morand C, Rémésy C, Jiménez L. (2004) Polyphenols: food
sources and bioavailability. Am J Clin Nutr;79: 727-47.
25. Duchnowicz P, Broncel M, Podsędek A, Koter-Michalak M. (2012) Hypolipidemic and
antioxidant effects of hydroxycinnamic acids, quercetin, and cyanidin 3-glucoside in
hypercholesterolemic erythrocytes (in vitro study). Eur J Clin Nutr;51: 435-43.
26. Roleira FM, Tavares-da-Silva EJ, Varela CL, Costa SC, Silva T, Garrido J, et al. (2015)
Plant derived and dietary phenolic antioxidants: Anticancer properties. Food Chem;183: 235-
58.
27. Razzaghi-Asl N, Garrido J, Khazraei H, Borges F, Firuzi O. (2013) Antioxidant
properties of hydroxycinnamic acids: a review of structure- activity relationships. Curr Med
Chem;20: 4436-50.
28. Zhao J, Cheng YY, Fan W, Yang CB, Ye SF, Cui W, et al. Botanical drug puerarin
coordinates with nerve growth factor in the regulation of neuronal survival and neuritogenesis
via activating ERK1/2 and PI3K/Akt signaling pathways in the neurite extension process.
CNS Neurosci Ther;21: 61-70.
29. Hoppe JB, Coradini K, Frozza RL, Oliveira CM, Meneghetti AB, Bernardi A, et al.
(2013) Free and nanoencapsulated curcumin suppress β-amyloid-induced cognitive
impairments in rats: involvement of BDNF and Akt/GSK-3β signaling pathway.
Neurobiology of learning and memory;106: 134-44.
30. Lv Q-Q, Wu W-J, Guo X-L, Liu R-L, Yang Y-P, Zhou D-S, et al. (2014) Antidepressant
activity of astilbin: involvement of monoaminergic neurotransmitters and BDNF signal
pathway. Biological and Pharmaceutical Bulletin;37: 987-95.
31. Zhang Z, Cai L, Zhou X, Su C, Xiao F, Gao Q, et al. (2015) Methyl 3, 4-
dihydroxybenzoate promote rat cortical neurons survival and neurite outgrowth through the
adenosine A2a receptor/PI3K/Akt signaling pathway. NeuroReport;26: 367-73.
32. Gaspar A, Martins M, Silva P, Garrido EM, Garrido J, Firuzi O, et al. (2010) Dietary
phenolic acids and derivatives. Evaluation of the antioxidant activity of sinapic acid and its
alkyl esters. J Agric Food Chem;58: 11273-80.
33. Esteves M, Siquet C, Gaspar A, Rio V, Sousa JB, Reis S, et al. (2008) Antioxidant versus
cytotoxic properties of hydroxycinnamic acid derivatives - a new paradigm in phenolic
research. Arch Pharm (Weinheim);341: 164-73.
34. Silva T, Bravo J, Summavielle T, Remião F, Pérez C, Gil C, et al. (2015) Biology-
oriented development of novel lipophilic antioxidants with neuroprotective activity. RSC
Adv;5: 15800-11.
35. Garrido J, Gaspar A, Garrido EM, Miri R, Tavakkoli M, Pourali S, et al. (2012) Alkyl
esters of hydroxycinnamic acids with improved antioxidant activity and lipophilicity protect
PC12 cells against oxidative stress. Biochimie;94: 961-7.
36. Andrade M, Benfeito S, Soares P, Magalhaes e Silva D, Loureiro J, Borges A, et al.
(2015) Fine-tuning of the hydrophobicity of caffeic acid: studies on the antimicrobial activity
against Staphylococcus aureus and Escherichia coli. RSC Adv;5: 53915-25.
This article is protected by copyright. All rights reserved.

37. Mosmann T. (1983) Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J Immunol Methods;65: 55-63.
38. Tavakkoli M, Miri R, Jassbi AR, Erfani N, Asadollahi M, Ghasemi M, et al. (2014)
Carthamus, Salvia and Stachys species protect neuronal cells against oxidative stress-induced
apoptosis. Pharm Biol;52: 1550-7.
39. Andrade M, Benfeito S, Soares P, e Silva DMe, Loureiro J, Borges A, et al. (2015) Fine-
tuning of the hydrophobicity of caffeic acid: studies on the antimicrobial activity against
Staphylococcus aureus and Escherichia coli. RSC Adv;5: 53915-25.
40. Menezes JC, Kamat SP, Cavaleiro JAS, Gaspar A, Garrido J, Borges F. (2011) Synthesis
and antioxidant activity of long chain alkyl hydroxycinnamates. Eur J Med Chem;46: 773-7.
41. Satoh T, Kosaka K, Itoh K, Kobayashi A, Yamamoto M, Shimojo Y, et al. (2008)
Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in
vivo through activation of the Keap1/Nrf2 pathway viaS-alkylation of targeted cysteines on
Keap1. J neuroch;104: 1116-31.
42. Ma Z-C, Hong Q, Wang Y-G, Tan H-L, Xiao C-R, Liang Q-D, et al. (2010) Ferulic acid
protects human umbilical vein endothelial cells from radiation induced oxidative stress by
phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways. Biol Pharm
Bull;33: 29-34.
43. Fukunishi Y, Mikami Y, Nakamura H. (2005) Similarities among receptor pockets and
among compounds: analysis and application to in silico ligand screening. J Mol Graph
Model;24: 34-45.
44. Ghorbani A, Sadeghnia HR, Asadpour E. (2015) Mechanism of protective effect of
lettuce against glucose/serum deprivation-induced neurotoxicity. Nutr Neurosci;18: 103-9.
45. Sang Zp, Qiang Xm, Li Y, Wu B, Zhang H, Zhao Mg, et al. (2015) Design, Synthesis,
and Biological Evaluation of Scutellarein Carbamate Derivatives as Potential Multifunctional
Agents for the Treatment of Alzheimer's Disease. Chem Biol Drug Des;86: 1168-77.
46. Tönges L, Ostendorf T, Lamballe F, Genestine M, Dono R, Koch JC, et al. (2011)
Hepatocyte growth factor protects retinal ganglion cells by increasing neuronal survival and
axonal regeneration in vitro and in vivo. J neurochem;117: 892-903
47. Gundimeda U, McNeill TH, Schiffman JE, Hinton DR, Gopalakrishna R. (2010) Green
tea polyphenols potentiate the action of nerve growth factor to induce neuritogenesis:
possible role of reactive oxygen species. J Neurosci Res;88: 3644-55.
48. Xu SL, Choi RC, Zhu KY, Leung K-W, Guo AJ, Bi D, et al. (2012) Isorhamnetin, a
flavonol aglycone from Ginkgo biloba L., induces neuronal differentiation of cultured PC12
cells: potentiating the effect of nerve growth factor. Evid Based Complement Alternat
Med;2012:
49. Nakajima K-i, Niisato N, Marunaka Y. (2011) Genistein enhances the NGF-induced
neurite outgrowth. Biomed Res;32: 351-6.
50. Teixeira J, Silva T, Benfeito S, Gaspar A, Garrido EM, Garrido J, et al. (2013) Exploring
nature profits: Development of novel and potent lipophilic antioxidants based on galloyl–
cinnamic hybrids. Eur J Med Chem;62: 289-96.
51. Hung C-C, Tsai W-J, Kuo L-MY, Kuo Y-H. (2005) Evaluation of caffeic acid amide
analogues as anti-platelet aggregation and anti-oxidative agents. Bioorg Med Chem;13: 1791-
7.
52. Kurauchi Y, Hisatsune A, Isohama Y, Mishima S, Katsuki H. (2012) Caffeic acid
phenethyl ester protects nigral dopaminergic neurons via dual mechanisms involving haem
oxygenase-1 and brain-derived neurotrophic factor. Br J Pharmaco;166: 1151-68.
53. dos Santos NA, Martins NM, Silva Rde B, Ferreira RS, Sisti FM, dos Santos AC. (2014)
Caffeic acid phenethyl ester (CAPE) protects PC12 cells from MPP+ toxicity by inducing the
expression of neuron-typical proteins. Neurotoxicology;45: 131-8.
This article is protected by copyright. All rights reserved.

54. Serafim TL, Carvalho FS, Marques MPM, Calheiros R, Silva T, Garrido J, et al. (2011)
Lipophilic caffeic and ferulic acid derivatives presenting cytotoxicity against human breast
cancer cells. Chem Res Toxicol;24: 763-74.
55. Sova M, Perdih A, Kotnik M, Kristan K, Rižner TLn, Solmajer T, et al. (2006) Flavonoids and
cinnamic acid esters as inhibitors of fungal 17β-hydroxysteroid dehydrogenase: A synthesis, QSAR
and modelling study. Bioorg Med Chem;14: 7404-18.
56. Kuihua Z, Chunyang W, Cunyi F, Xiumei M. (2014) Aligned SF/P (LLA‐CL)‐blended
nanofibers encapsulating nerve growth factor for peripheral nerve regeneration. J Biomed
Mater Res A;102: 2680-91.
57. Zhang H, Wu F, Kong X, Yang J, Chen H, Deng L, et al. (2014) Nerve growth factor
improves functional recovery by inhibiting endoplasmic reticulum stress-induced neuronal
apoptosis in rats with spinal cord injury. J Transl Med;12: 130.
58. Bannwarth B, Kostine M. (2014) Targeting nerve growth factor (NGF) for pain
management: what does the future hold for NGF antagonists? Drugs;74: 619-26.
59. Hetman M, Gozdz A. (2004) Role of extracellular signal regulated kinases 1 and 2 in
neuronal survival. Eur J Biochem;271: 2050-5.
60. Longo FM, Massa SM. (2013) Small-molecule modulation of neurotrophin receptors: a
strategy for the treatment of neurological disease. Nat Rev Drug Discov;12: 507-25.
61. Nguyen CN, Kim HE, Lee SG. (2013) Caffeoylserotonin Protects Human Keratinocyte
HaCaT Cells against H2O2‐Induced Oxidative Stress and Apoptosis through Upregulation of
HO‐1 Expression via Activation of the PI3K/Akt/Nrf2 Pathway. Phytother Res;27: 1810-8.
62. Chai L, Guo H, Li H, Wang S, Wang Y-l, Shi F, et al. Scutellarin and caffeic acid ester
fraction, active components of Dengzhanxixin injection, upregulate neurotrophins synthesis
and release in hypoxia/reoxygenation rat astrocytes. J Ethnopharmacol;150: 100-7.
63. Huang Y, Jin M, Pi R, Zhang J, Chen M, Ouyang Y, et al. (2013) Protective effects of
caffeic acid and caffeic acid phenethyl ester against acrolein-induced neurotoxicity in HT22
mouse hippocampal cells. Neurosci Lett;535: 146-51.
64. Zhang H, Mak S, Cui W, Li W, Han R, Hu S, et al. (2011) Tacrine (2)–ferulic acid, a
novel multifunctional dimer, attenuates 6-hydroxydopamine-induced apoptosis in PC12 cells
by activating Akt pathway. Neurochem Int;59: 981-8.
65. Jin Y, FAN Y, LIU Z. (2007) Neuroprotection by sodium ferulate against
glutamate‐induced apoptosis is mediated by ERK and PI3 kinase pathways1. Acta Pharmacol
Sin;28: 1881-90.
66. Jin Y, FAN Y, LI Z. (2005) Sodium ferulate prevents amyloid‐beta‐induced neurotoxicity
through suppression of p38 MAPK and upregulation of ERK‐1/2 and Akt/protein kinase B in
rat hippocampus1. Acta Pharmacol Sin;26: 943-51.
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Table 1. Caffeic acid and its alkyl esters enhance NGF-induced neurite outgrowth in
PC12 cells.
Compound
Code
Concentration
(µM)*
Number of neurites Number
neurites
of
(d ≥ 100 µm)
1
2.5 2.5
0.5 0.5
5
25
10.3 10.3
11.7 7.5
0.3 0.5
1.2 0.4
CAF
1
5
25
6.0 3.0
1.2 0.2
9.0a 4.2
1.8 1.2
50.1ab 18.5
38.3a 9.3
CAF1
CAF2
CAF3
CAF4
CAF5
1
5
25
11.9 0.1
2.1 0.1
13.7ab 8.7
2.0 2.0
66.9ab 37.6
36.7a 18.8
O
1
5
25
21.4 3.4
88.1ab 38.9
26.2 9.8
4.7 1.3
17.7ab 7.8
2.3 1.2
HO
HO
OCH2CH2CH3
1
5
25
21.3 4.3
78.0ab 18.8
15.9 4.1
7.3 1.6
17.0ab 3.0
3.8 1.1
1
5
25
6.5 1.5
14.2 2.9
3.0 3.0
0.5 0.5
2.3 0.7
0.7 0.0
5 ng/mL
50 ng/mL
NGF
NGF
0.5 0.3
16.3 0.8
0.5 0.1
2.6 0.2
* NGF 5 ng/mL was used in each assay. a Significantly different from cells treated with NGF
5 ng/mL (P<0.05).b Significantly different from cells treated with NGF 50 ng/mL (P<0.05)
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Table 2. Docking validation results for PDB structure of PI3K (1E7U) using AutoDock
4.2
PDB
code
Binding mode of wortmannin with PI3K active
site
No. GA Population in
RMSD from
reference
structure(°A)
Estimated free
energy of binding
(kcal/Mol)
runs
the optimum
cluster (%)
1E7U
100
80
1.74
-8.96
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