Serotonin derivatives as inhibitors of β-secretase (BACE 1)

Article abstract of DOI:10.1691/ph.2011.0789

All serotonin derivatives described here (1-9) inhibited BACE 1 in a dose dependent manner. The 50% Inhibition Concentration (IC₅₀) of N-cinnamoyl serotonin (1) was 86.7 ± 4.0 μM. The peptide conjugation of serotonin derivatives influenced the BACE 1 inhibitory activity. Among serotonin derivatives (1-8), introduction of substituents, such as hydroxyl and methoxy groups at the 4′-position decreased the inhibitory activity (N-p-coumaroyl serotonin (2), N-p-methoxy cinnamoyl serotonin (3)). With a hydroxylgroup at the 4′-position, and the meta-hydroxy function being substituted by a hydroxyl group or methoxy group (Ncaffeoyl serotonin (4), N-feruloyl serotonin (5)), inhibitory activity was weakened, (IC₅₀ > 400 μM). BACE 1 inhibitory activity was effected by the substituents of the cinnamic acid moiety. This is the first report on Structure-Activity- Relationships (SAR) for the BACE 1-inhibiting activity of serotonin derivatives. These serotonin derivatives, which have anti-oxidative effects as well are expected to be useful in the study of the mechanisms of Alzheimer's disease.

Full text of DOI:10.1691/ph.2011.0789

ORIGINAL ARTICLES
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Osaka, Japan
Serotonin derivatives as inhibitors of ␤-secretase (BACE 1)
T. Takahashi, M. Miyazawa
Received September 21, 2010, accepted October 22, 2010
Prof. Mitsuo Miyazawa, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka-shi,
Osaka 577-8502, Japan
miyazawa@kindai.ac.jp
Pharmazie 66: 301–305 (2011)
doi: 10.1691/ph.2011.0789
All serotonin derivatives described here (1–9) inhibited BACE 1 in a dose dependent manner. The 50%
Inhibition Concentration (IC50) of N-cinnamoyl serotonin (1) was 86.7 4.0 ␮M. The peptide conjugation
of serotonin derivatives influenced the BACE 1 inhibitory activity. Among serotonin derivatives (1–8), intro-
duction of substituents, such as hydroxyl and methoxy groups at the 4ꢀ-position decreased the inhibitory
activity (N-p-coumaroyl serotonin (2), N-p-methoxy cinnamoyl serotonin (3)). With a hydroxylgroup at the
4ꢀ-position, and the meta-hydroxy function being substituted by a hydroxyl group or methoxy group (N-
caffeoyl serotonin (4), N-feruloyl serotonin (5)), inhibitory activity was weakened, (IC50 > 400 ␮M). BACE
1 inhibitory activity was effected by the substituents of the cinnamic acid moiety. This is the first report
on Structure-Activity-Relationships (SAR) for the BACE 1-inhibiting activity of serotonin derivatives. These
serotonin derivatives, which have anti-oxidative effects as well are expected to be useful in the study of the
mechanisms of Alzheimer’s disease.
1. Introduction
of the antioxidant potential of food which would therefore act
as a natural source of antioxidants (Parr and Bolwell 2000). In
the sense, polyphenols might be a good candidate for BACE 1
inhibitors, however, natural product inhibitors have rarely been
investigated (Jeou et al. 2003; Choi et al. 2008; Marumoto and
Miyazawa 2010). Thus, we focused our interest to antioxidant
substances from natural sources.
Serotonin derivatives were identified as the major unique phe-
nolic compounds of safflower seeds, belonging to a family of
indole hydroxyl cinnamic acid amides (Sakamura et al. 1978;
Sato et al. 1985). These compounds have a number of biological
effects, such as, antioxidative activities (Zhang et al. 1997), anti-
tyrosinase activity, melanin-production inhibitory activity (Roh
et al. 2004), anti-tumor activity (Nagatsu et al. 2000), fibrob-
lasts growth promoting activities (Takii et al. 1999). Among the
serotonin derivatives, N-p-coumaroyl serotonin and N-feruloyl
serotonin are commomly detected, while only few other sero-
tonin derivatives have been reported (Park 2008; Yamazaki et al.
2009; Kang et al. 2009).
Alzheimer’s disease (A. D) is a meurodegerative diorder dini-
cally characterized by progressive dementia that inevitably leads
to incapacitation and death. Althoug the pathogenesis of AD
is complicated and involves in numerous pathways, two major
hypotheses are currently under considaration regarding the
molecular mechanism, the cholinergic hypothesis and the amy-
loidcascadehypothesis. Thus, thefocushereinisuponinhibitors
of select cholinesterase (ChEs) to alleviate cholinergic deficits
and improve neurotransmission and ␤-siteamyloid precursor
protein (APP) cleaving enzyme 1 (BACE 1; as party protease,
␤-secretase, and memapsin 2) inhibitors to preclude formation
and accumulation of amyloid ␤ peptide (A␤)· Pursuant to this,
both could then be established as visable therapeutic targets for
AD. Furthermore, recent evidence in the field of Alzheimer’s
disease research has highlighted the importance of the oxida-
tive process in its pathogenesis. Based on laboratory and clinical
studies, itappearsthatreactiveoxygenspeciesandreactivenitro-
gen species that are generated extracellulary and intracellulary
by various mechanisms are among the major intermediary risk
factors that initiate and promote neuodegeration in idiopathic
AD(Prasadetal. 2000, 2002). Thus, attenuationofoxidantstress
by antioxidant molecules is a potential therapeutic intervention
in AD. In order to have therapeutic potential, the molecular
weight of inhibitors should be below 700 Da, because efficacy
requires penetration of the blood-brain barrier. For this reason,
large peptide-based inhibitors are not proper drug candidates,
butthesecondarymetabolitesofplantswhichhaverelativelylow
molecular weight and high lipophilicity might be good BACE 1
inhibitors (Dorrvel 2000; Stachel et al. 2004).
In this study, we synthesized a series of serotonin derivatives
(Fig. 1) using cinnamic acid derivatives, and evaluated their
antioxidant ␤−secretase inhibitory effects.
2. Investigations, results and discussion
2.1. Antioxidant activities of serotonin derivatives 1–9
The antioxidant activities of compounds 1–9 were evaluated by
EC50 (50% Effective Concentration) in DPPH radical scaveng-
ing activity as shown in Fig. 2.
All serotonin derivatives 1–9 were found to be stronger than
BHT in free radical scavenging activity. Therefore, in order
to clarify whether the antioxidant activity of serotonin deriva-
tives was due to the cinnamic acid or the serotonin moiety, we
evaluated the antioxidant activities of cinnamic acid derivatives.
Polyphenols are group of phytochemicals that exhibit a wide
range of physiological and therapeutic properties, including
anti-allergenic, anti-inflammatory, anti-microbial and antioxi-
dant effects. Phenolic compounds could be a major determinant
Pharmazie 66 (2011)
301

ORIGINAL ARTICLES
Fig. 1: Structure of serotonin derivatives (1–9)
Each serotonin derivative showed a stronger antioxidant activ-
ity than the cinnamic acid derivatives (Table 1). Furthermore,
the EC50 value of bufobutanoic acid (9) which did not conju-
gate with the cinnamic acid moiety was 43.6 2.1 ␮M. These
results indicated that the antioxidant activity is strengthened by
the serotonin moiety. These results supported previous sugges-
tions (Takii et al. 1999). In the case of cinnamic acid derivatives,
the substituents of cinnamic acid derivatives effect antioxidant
activity, whereas the substituents of serotonin derivatives appear
to be less important. The serotonin moiety effected the radical
scavenging activity strongly.
2.2. β-Secretase inhibitory effects of serotonin derivatives
1–9 and Structure-Activity-Relationships (SAR)
All the serotonin derivatives 1–9 inhibited BACE 1 in a dose
dependent manner (Fig. 3), in particular N-cinnamoyl serotonin
(1) which has no substituents of cinnamic acid. The 50%
100
1
2
80
3
100
1
4
2
3
80
60
40
20
0
5
4
5
6
7
8
9
6
60
7
40
8
9
20
BHT
0
0
100
200
300
400
500
0
50
100
150
200
Concentration (µM)
µ
Concentration ( M)
Fig. 3: ␤–Secretase inhibitory effects of serotonin derivatives (1–9)
Fig. 2: DPPH radical scavenging activities of serotonin derivatives (1–9)
Table 1: EC50 values of serotonin derivatives 1–9 and cinnamic acid derivatives
Compound
Ec (␮M)
50
Compound
Ec (␮M)
50
N-Cinnamoyl serotonin (1)
N-p-Coumaroyl serotonin (2)
N-p-Methoxy-cinnamoyl serotonin (3)
N-Caffeoyl serotonin (4)
N-Feruloyl serotonin (5)
N-Iso feruloyl serotonin (6)
N-3,4-Dimethoxy cinnamoyl serotonin (7)
N-Sinapoyl serotonin (8)
Bufobutanoic acid (9)
20.9 2.3
21.4 1.9
36.8 2.1
18.9 2.0
20.2 2.1
39.2 2.2
30.5 2.4
27.2 2.2
43.6 2.1
104.8 2.0
Cinnamic acid
>250
>250
>250
155.3 3.2
>250
>250
>250
>250
p-Coumaric acid
p-Methoxy cinnamic acid
Caffeic acid
Ferulic acid
Isoferulic acid
3,4-Dimethoxy cinnamic acid
Sinapic acid
BHT*
*
reference compound
The results are the means SD of three experiments
302
Pharmazie 66 (2011)

ORIGINAL ARTICLES
Table 2: ␤-Secretase inhibitory activity of serotonin derivatives 1–9
Compound
Concentration (␮M)
Inhibition (%)
IC (␮M)
50
Compound
Concentration (␮M)
Inhibition (%)
IC (␮M)
50
1
25
100
400
25
100
400
25
100
400
25
100
400
25
29.3 3.2
52.5 3.5
29.3 5.2
12.9 1.3
32.7 4.1
65.0 5.7
0.2 0.1
6
25
100
400
25
100
400
25
100
400
25
100
400
25
0.0 0.4
11.8 3.2
26.1 4.0
0.4 0.5
9.3 0.7
41.3 2.6
3.2 1.2
26.6 3.1
73.3 3.9
19.5 0.2
46.8 1.0
84.6 2.5
86.7 4.0
210.8 10.3
>400
>400
2
3
4
5
7
>400
8
28.2 2.3
31.8 2.1
200.3 6.8
110.6 4.0
>400
0
0.2
9
5.1 1.2
35.3 2.1
0.0 0.2
5.1 1.2
35.3 2.1
>400
Cinnamic acid
p-Coumaric acid
reference*
0
1.0
100
400
100
400
25
100
400
0.9 0.5
3.2 0.9
>400
0
1.0
4.7 2.2
16.7 3.1
>400
0.2 0.01
*
Lys-Thr-Glu-lle-Ser-Glu-Val-Asn-(statine)-Val-Ala-Glu-Phe-OH was used as the positive control
The results are the means SD of three experiments
Inhibition Concentration (IC50) of 1 was 86.7 4.0 ␮M. In
order to clarify whether the ␤-secretase inhibitory activity
of serotonin derivatives was due to the cinnamic acid or
serotonin moiety, we evaluated BACE 1 inhibitory activities
of cinnamic acid and p-coumaric acid. Since the inhibitory
activities of cinnamic acid and p-coumaric acid were lower
than that of serotonin derivatives, furthermore, bufobutanoic
acid which did not have a cinnamic acid moiety showed BACE
1 inhibitory activity (IC50 = 110.6 4.0 ␮M). We assumed that
peptide conjugation of serotonin derivatives has effects on
BACE 1. Among the serotonin derivatives 1–8, introduction
of substituents, such as hydroxyl and methoxy groups at the
4ꢀ-position decreased the inhibitory activity (N-p-coumaroyl
serotonin (2), N-p-methoxy cinnamoyl serotonin (3). When
a hydroxylgroup was located at the 4ꢀ-position, and the
meta-hydroxy function was substituted by a hydroxyl group or
methoxy group (N-caffeoyl serotonin (4), N-feruloyl serotonin
(5)), inhibitory activities were weakened, (IC50 > 400 ␮M).
Furthermore, the introduction of another methoxy group in
a ortho-position to a hydroxyl group N-sinapoyl serotonin
(8) lead to an increase in the inhibitory activity relatively to
N-feruloyl serotonin. When a methoxy group was located at the
4ꢀ-position, and the meta-methoxy function was substituted by
a hydroxyl group or methoxy group a (N-isoferuloyl serotonin
(6), N-3ꢀ, 4ꢀ-dimethoxy cinnamoyl serotonin (7)) also inhibitory
activity was weakened (IC50 > 400) (Table 2).
raphy was carried out using 70–230 mesh silica gel (Kieselgel 60, Merck,
Germany). Melting points (m.p.) were measured on a Yanaco MP-5000D
melting-point apparatus. UV spectra were measured on a Hitachi U-1500
spectrophotometer. 1Hand13CNMRdatawereallobtainedonaJEOLECA-
400 (1H: 400 MHz; 13C: 100 MHz), JEOL ECA-500 (1H: 500 MHz; 13C:
125 MHz) and JEOL ECA-700 (1H: 700 MHz; 13C: 175 MHz) in DMSO-d6
with TMS as internal standard. IR spectra were obtained with a Jasco FT/IR-
470 plus Fourier transform infrared spectromoter. The FAB-MS spectrum
was obtained with a Jeol Tandem MS station JMS-700TKM. Fluorescence
was measured with a MTP-800Lab microplate fluorescence reader. Sero-
tonin hydrochloride, cinnamic acid, p-coumaric acid, ferulic acid, caffeic
acid, sinapic acid, p-methoxy cinnamic acid, 3,4-dimethoxy cinnamic acid,
DPPH (1,1-diphenyl-2-picrylhydrazyl), BHT (2,6-di-tert-butyl-4-methyl-
phenol), succinic anhydride, piperidine, HOBT (1-hydroxybenzotriazole),
tetrakis(triphenylphosphine)palladium morpholine, (0) EDC (1-ethyl-3-
(3-dimethylaminopropyl)-carbodiimide) were purchased from Wako Pure
Chemistry (Osaka, Japan), 3,4-dihydroxy denzaldehyde and dimethy-
laminopyridine were purchased from Tokyo Kasei Kogyo (Tokyo, Japan).
Isoferulic acid and all solvents were purchased from Kanto Chemical
(Tokyo, Japan).
3.2. Synthesis of serotonin derivatives 1–3, 5–8
Synthesis of serotonin derivatives 1–3, 5–8 was accomplished accord-
ing to a published method (Koyama et al. 2006). First, triethylamine
(230 ␮l, 1.65 mmol), 1-hydroxybenzotriazole (223 mg, 1.65 mmol) and
EDC (290 ␮l, 1.65 mmol) were added to a solution of serotonin hydrochlo-
ride (350 mg, 1.65 mmol) and cinnamic acid derivatives (1.50 mmol) in
dimethylformamide(1.5 ml) and dichloromethane (10 ml). Afterthemixture
was stirred over night at room temperature under nitrogen, it was concen-
trated in vacuo. The residue was treated with water (15 ml) and extracted
with ethyl acetate (3 × 10 ml). The organic extract was washed successively
with 5% citric acid solution (3 × 20 ml), saturated sodium hydrogencar-
bonate solution (3 × 30 ml), and brine (50 ml). The extract was dried over
Na2SO4 and concentrated to yield a solid (yield 64.9%–96.1%).
BACE 1 inhibitory activity was effected by the substituents
of the cinnamic acid moiety. The results were summarized in
Table 2. These serotonin derivatives which have relatively low
molecular weight might not be drug candidates themselves since
their inhibitory activity is not strong. However, this is the first
report on Structure-Activity-Relationships (SAR) for the BACE
1-inhibiting activity of serotonin derivatives. These serotonin
derivatives, which have anti-oxidative effects as well as BACE
1-inhibitory activity are expected to be useful in the study of the
mechanisms of Alzheimer’s disease.
3.2.1. N-Cinnamoyl serotonin (1)
Violet solid: Yield 95.0%; mp 92–95 ◦C. IR ␯
(KBr): 3292, 1654, 1607,
max
1531, 798 cm−1. 1H NMR (400 MHz, DMSO-d6) ␦ 10.48 (1H, s, H-1), 8.50
(1H, s, 5-OH), 8.19 (1H, t, 5.6 Hz, -CONH), 7.55 (2H, d, 6.8 Hz, H-2ꢀ, and
H-6ꢀ), 7.42 (1H, d, 16.0 Hz, H-7ꢀ), 7.38 (3H, m, H-3ꢀ, H-4ꢀ and H-5ꢀ), 7.12
(1H, d, 8.0 Hz, H-7), 7.05 (1H, s, H-2), 6.85 (1H, d, 2.4 Hz, H-4), 6.63 (1H,
d, 16.0 Hz, H-8ꢀ), 6.56 (1H, dd, 8.0 Hz, 2.4 Hz, H-6), 3.44 (2H, dt, 5.6 Hz,
6.0 Hz, H-11), 2.79 (2H, t, 6.0 Hz, H-10). 13C NMR (100 MHz, DMSO- d6)
␦ 164.8 (C-9ꢀ), 150.2 (C-5), 138.4 (C-7ꢀ), 135.0 (C-1ꢀ), 130.8 (C-8), 129.3
(C-4ꢀ), 128.9 (C-2ꢀ and C-6ꢀ), 127.9 (C-9), 127.5 (C-3ꢀ and C-5ꢀ), 123.1 (C-
2), 122.4 (C-8ꢀ), 111.6 (C-7), 111.3 (C-6), 110.8 (C-3), 102.2 (C-4), 39.5
(C-11), 25.4 (C-10).
3. Experimental
3.1. Materials
Thin layer chromatography (TLC) was performed on precoated plates (silica
gel 60 F254, 0.25 mm, Merck, Darmstadt, Germany). Column chromatog-
Pharmazie 66 (2011)
303

ORIGINAL ARTICLES
3.2.2. N-p-Coumaroyl serotonin (2)
3.42 (2H, dt, 4.7 Hz, 7.2 Hz, H-11), 2.79 (2H, t, 7.2 Hz, H-10). 13C NMR
(100 MHz, DMSO- d6) ␦ 165.3 (C-9ꢀ), 150.2 (C-5), 148.0 (C-3ꢀ and C-5ꢀ),
139.1 (C-7ꢀ), 137.2 (C-4ꢀ), 130.8 (C-8), 127.9 (C-9), 125.4 (C-1ꢀ), 123.1
(C-2), 119.6 (C-8ꢀ), 111.6 (C-7), 111.3 (C-6), 110.8 (C-3), 105.2 (C-2ꢀ and
C-6ꢀ), 102.2 (C-4), 55.9 (3ꢀ-OMe and 5ꢀ-OMe), 39.4 (C-11), 25.4 (C-10).
Violet solid: Yield 75.0%; mp 192–195 ◦C. FAB-MS m/z 323 [M + H]+.
IR ␯
(KBr): 3318, 1586, 1512, 830 cm−1. 1H NMR (500 MHz, DMSO-
max
d6) ␦ 10.48 (1H, d, 2.3 Hz, H-1), 8.60 (1H, s, 5-OH), 8.06 (1H, t, 5.6 Hz,
-CONH), 7.38 (2H, d, 8.6 Hz, H-2ꢀ and H-6ꢀ), 7.33 (1H, d, 16.0 Hz, H-7ꢀ),
7.12 (1H, d, 8.6 Hz, H-7), 7.05 (1H, d, 2.3 Hz, H-2), 6.85 (1H, d, 2.3 Hz,
H-4), 6.79 (2H, d, 8.6 Hz, H-3ꢀ and H-5ꢀ), 6.59 (1H, dd, 8.6 Hz, 2.3 Hz, H-6),
6.41 (1H, d, 16.0 Hz, H-8ꢀ), 3.42 (2H, dt, 5.6 Hz, 7.8 Hz, H-11), 2.78 (2H,
t, 7.8 Hz, H-10). 13C NMR (125 MHz, DMSO- d6) ␦ 165.5 (C-9ꢀ), 159.0
(C-4ꢀ), 150.4 (C-5), 138.7 (C-7ꢀ), 131.0 (C-8), 129.4 (C-2ꢀ and C-6ꢀ), 128.1
(C-9), 126.2 (C-1ꢀ), 123.3 (C-2), 119.1 (C-8ꢀ), 115.9 (C-3ꢀ and C-5ꢀ), 111.8
(C-7), 111.5 (C-6), 111.0 (C-3), 102.4 (C-4), 40.0 (C-11), 25.6 (C-10).
3.3. Synthesis of N-caffeoyl serotonin (4)
First, to a solution of 3,4-dihydroxy benzaldehyde (297 mg, 2.15 mmol) in
anh. dimethylformamide (3.0 ml) was added allyl bromide (1.2 equiv. per
OH-group) and K2CO3 (1.2 equiv.) and NaI (90 mg, 0.60 mmol), the mixture
was heated at 60 ◦C for 2 h. The mixture was treated with 5% HCl solution,
and extracted with ethyl acetate (10 ml × 3). The organic extract was washed
successively with water (3 × 10 ml) and brine (50 ml). The extract was dried
over Na2SO4 and concentrated to obtained 3,4-diallyloxybenzaldehyde
(459.3 mg, yield 98.0%). Secondly, 3,4-diallyloxybenzaldehyde and mal-
onic acid were dissolved in anh. pyridine (2 ml/mmol aldehyde) containing
3 vol% of piperidine. The mixture was stirred under reflux for 2 h,
cooled and poured into ice-cold 2N-HCl. The precipitate was filtered and
taken up in EtOAc. The organic layer was washed with 2 N HCl, dried
(Na2SO4) and concentrated to give 3,4-di-O-allylcaffeic acid (453.7 mg,
yield 82.6%). Triethylamine (230 ␮l, 1.65 mmol), 1-hydroxybenzotriazole
(223 mg, 1.65 mmol) and EDC (290 ␮L, 1.65 mmol) were added to a
solution serotonin hydrochloride (350 mg, 1.65 mmol) and compound 4ꢀꢀ
(1.50 mmol) in dimethylformamide (1.5 ml) and dichloromethane (10 ml).
After the mixture was stirred over night at room temperature under nitro-
gen, it was concentrated in vacuo. The residue was treated with water
(15 ml) and extracted with ethyl acetate (3 × 10 ml). The organic extract
was washed successively with 5% citric acid solution (3 × 20 ml), satu-
rated sodium hydrogencarbonate solution (3 × 30 ml), and brine (50 ml).
The extract was dried over Na2SO4 and concentrated to yield a solid (yield
96.3%). The ally protected compound 4 was dissolved in degassed anh. THF
(50 ml/mmol) and morpholine (10 equiv. per allyl group to be cleaved) was
added Pd (PPh3)4 (1 mol%). The green mixture was stirred at room tempera-
ture (monitored by TLC) and concentrated in vacuo. The residue was treated
with saturated NH4Cl solution and extracted with ethyl acetate (3 × 10 ml).
The extract was dired over Na2SO4 and concentrated to yield a pale yellow
solid. The extract was separated by silica gel column chromatography with a
hexane-EtOAc (1: 1, v/v) system to give N-caffeoyl serotonin (4) (422.0 mg,
yield 83.3%).
3.2.3. N-p-Methoxy cinnamoyl serotonin (3)
Pale yellow solid: Yield 79.2%; mp 178–185 ◦C. IR ␯max (KBr): 3271, 1650,
1601, 1574, 1509, 829 cm−1. 1H NMR (400 MHz, DMSO- d6) ␦ 10.50 (1H,
d, 2.3 Hz, H-1), 8.12 (1H, t, 6.0 Hz, -CONH), 7.51 (2H, d, 8.8 Hz, H-2ꢀ and
H-6ꢀ), 7.39 (1H, d, 15.6 Hz, H-7ꢀ), 7.13 (1H, d, 8.8 Hz, H-7), 7.06 (1H, d,
2.3 Hz, H-2), 6.86 (1H, d, 2.0 Hz, H-4), 6.76 (2H, d, 8.8 Hz, H-3ꢀ and H-5ꢀ),
6.66 (1H, dd, 8.8 Hz, 2.0 Hz, H-6), 6.50 (1H, d, 15.6 Hz, H-8ꢀ), 3.78 (3H,
s, 4ꢀ-OMe), 3.44 (2H, dt, 6.0 Hz, 7.8 Hz, H-11), 2.79 (2H, t, 7.8 Hz, H-10).
13C NMR (100 MHz, DMSO- d6) ␦ 165.1 (C-9ꢀ), 160.2 (C-4ꢀ), 150.2 (C-5),
138.1 (C-7ꢀ), 130.8 (C-8), 129.0 (C-2ꢀ and C-6ꢀ), 127.9 (C-9), 127.5 (C-1ꢀ),
123.1 (C-2), 119.9 (C-8ꢀ), 114.4 (C-3ꢀ and C-5ꢀ), 111.6 (C-7), 111.3 (C-6),
110.8 (C-3), 102.2 (C-4), 55.2 (4ꢀ-OMe) 39.4 (C-11), 25.4 (C-10).
3.2.4. N-Feruloyl serotonin (5)
Pale violet solid: Yield 96.1%; mp 110–114 ◦C. IR ␯max (KBr): 3374, 1652,
1589, 1514, 844 cm−1 1H NMR (500 MHz, DMSO- d6) ␦ 10.49 (1H, d,
.
2.0 Hz, H-1), 9.42 (1H, s, 4ꢀ-OH), 8.61 (1H, s, 5-OH), 8.05 (1H, t, 5.8 Hz,
-CONH), 7.34 (1H, d, 15.6 Hz, H-7ꢀ), 7.13 (1H, d, 8.6 Hz, H-7), 7.12 (1H, d,
2.0 Hz, H-2ꢀ), 7.06 (1H, d, 2.0 Hz, H-2), 6.99 (1H, dd, 8.1 Hz, 2.0 Hz, H-6ꢀ),
6.86 (1H, d, 2.3 Hz, H-4), 6.79 (1H, d, 8.1 Hz, H-5ꢀ), 6.60 (1H, dd, 8.6 Hz,
2.3 Hz, H-6), 6.46 (1H, d, 15.6 Hz, H-8ꢀ), 3.81 (3H, s, 3ꢀ-OMe), 3.43 (2H,
dt, 5.8 Hz, 7.5 Hz, H-11), 2.78 (2H, t, 7.5 Hz, H-10). 13C NMR (125 MHz,
DMSO- d6) ␦ 165.5 (C-9ꢀ), 150.4 (C-5), 148.4 (C-4ꢀ), 148.0 (C-3ꢀ), 139.0
(C-7ꢀ), 131.0 (C-8), 128.1 (C-9), 126.7 (C-1ꢀ), 123.3 (C-2), 121.7 (C-6ꢀ),
119.4 (C-8ꢀ), 115.8 (C-5ꢀ), 111.9 (C-7), 111.5 (C-6), 111.0 (C-3), 110.9
(C-2ꢀ), 102.4 (C-4), 55.7 (3ꢀ-OMe), 39.6 (C-11), 25.6 (C-10).
Pale yellow solid: Yield 64.9%; mp 98–102 ◦C. IR ␯max (KBr): 3409, 1647,
1575, 1538, 811 cm−1 1H NMR (400 MHz, DMSO- d6) ␦ 10.51 (1H, s,
.
H-1), 8.14 (1H, t, 5.2 Hz, -CONH), 7.27 (1H, d, 15.6 Hz, H-7ꢀ), 7.14 (1H,
d, 8.0 Hz, H-7), 7.07 (1H, s, H-2), 6.98 (1H, d, 2.0 Hz, H-2ꢀ), 6.87 (1H, d,
2.0 Hz, H-4), 6.85 (1H, dd, 2.0 Hz, 8.0 Hz, H-6ꢀ) 6.77 (1H, d, 8.0 Hz, H-5ꢀ),
6.61 (1H, dd, 8.0 Hz, 2.0 Hz, H-6), 6.37 (1H, d, 15.6 Hz, H-8ꢀ), 3.43 (2H,
dt, 5.2 Hz, 7.6 Hz, H-11), 2.79 (2H, t, 7.6 Hz, H-10). 13C NMR (100 MHz,
DMSO- d6) ␦ 165.4 (C-9ꢀ), 150.2 (C-5), 147.3 (C-4ꢀ), 145.6 (C-3ꢀ), 139.0
(C-7ꢀ), 130.8 (C-8), 127.9 (C-9), 126.5 (C-1ꢀ), 123.1 (C-2), 120.4 (C-6ꢀ),
118.8 (C-8ꢀ), 115.8 (C-5ꢀ), 113.9 (C-2ꢀ), 111.7 (C-7), 111.3 (C-6), 110.9
(C-3), 102.3 (C-4), 39.5 (C-11), 25.5 (C-10).
3.2.5. N-Isoferuloyl serotonin (6)
Pale violet solid: Yield 77.0%; mp 99–105 ◦C. IR ␯
(KBr): 3390, 1652,
max
1584, 1509, 801 cm−1 1H NMR (400 MHz, DMSO- d6) ␦ 10.47 (1H, d,
.
2.0 Hz, H-1), 9.16 (1H, s, 3ꢀ-OH), 8.59 (1H, s, 5-OH), 8.10 (1H, t, 6.0 Hz,
-CONH), 7.27 (1H, d, 15.6 Hz, H-7ꢀ), 7.10 (1H, d, 8.0 Hz, H-7), 7.04 (1H, d,
2.0 Hz, H-2), 6.97 (1H, d, 1.2 Hz, H-2ꢀ), 6.95 (1H, dd, 8.4 Hz, 1.2 Hz, H-6ꢀ),
6.92 (1H, d, 8.4 Hz, H-5ꢀ), 6.86 (1H, d, 1.6 Hz, H-4), 6.58 (1H, dd, 8.0 Hz,
1.6 Hz, H-6), 6.39 (1H, d, 15.6 Hz, H-8ꢀ), 3.78 (3H, s, 4ꢀ-OMe), 3.41 (2H,
dt, 6.0 Hz, 7.2 Hz, H-11), 2.77 (2H, t, 7.2 Hz, H-10). 13C NMR (100 MHz,
DMSO- d6) ␦ 165.1 (C-9ꢀ), 150.2 (C-5), 149.1 (C-4ꢀ), 146.7 (C-3ꢀ), 138.5
(C-7ꢀ), 130.8 (C-8), 127.9 (C-9), 127.8 (C-1ꢀ), 123.1 (C-2), 120.1 (C-6ꢀ),
119.7 (C-8ꢀ), 113.3 (C-5ꢀ), 112.1 (C-7), 111.6 (C-6), 111.3 (C-3), 110.8
(C-2ꢀ), 102.4 (C-4), 55.6 (4ꢀ-OMe), 39.4 (C-11), 25.4 (C-10).
3.4. Synthesis of bufobutanoic acid (9)
Synthesis of bufobutanoic acid (9) was accomplished according to a pub-
lished method (Somei et al. 2001). Succinic anhydride (150 mg, 1.51 mmol)
was added to a solution of serotonin hydrochloride (353.1 mg, 1.66 mmol)
in DMF (15.0 ml) and pyridine (1.5 ml) at room temperature and stirring was
continued for 24 h. After evaporation under reduced pressure, the residual
oil was column chromatographed on SiO2 with CH2Cl2-MeOH-AcOH (10:
1: 0.1, v/v) to give bufobutanoic acid (9) (258.4 mg, 62.0%).
3.2.6. N-3,4-Dimethoxy cinnamoyl serotonin (7)
Pale violet solid: Yield 82.0%; mp 103–105 ◦C. IR ␯max (KBr): 3369, 1654,
1595, 1542, 802 cm−1 1H NMR (700 MHz, DMSO- d6) ␦ 10.49 (1H, s,
.
(KBr): 3294, 1652, 1558, 798 cm−1 1H
.
H-1), 8.07 (1H, t, 5.6 Hz, -CONH), 7.37 (1H, d, 15.6 Hz, H-7ꢀ), 7.15 (1H, d,
1.4 Hz, H-2ꢀ), 7.13 (1H, d, 8.7 Hz, H-7), 7.11 (1H, dd, 8.4 Hz, 1.4 Hz, H-6ꢀ),
7.07 (1H, s, H-2), 6.98 (1H, d, 8.4 Hz, H-5ꢀ), 6.86 (1H, d, 2.2 Hz, H-4),
6.60 (1H, dd, 8.7 Hz, 2.2 Hz, H-6), 6.53 (1H, d, 15.6 Hz, H-8ꢀ), 3.80 (3H, s,
3ꢀ-OMe), 3.78 (3H, s, 4ꢀ-OMe), 3.44 (2H, dt, 5.6 Hz, 7.4 Hz, H-11), 2.79
(2H, t, 7.4 Hz, H-10). 13C NMR (175 MHz, DMSO- d6) ␦ 165.4 (C-9ꢀ),
150.4 (C-5), 150.2 (C-4ꢀ), 149.1 (C-3ꢀ), 138.7 (C-7ꢀ), 131.0 (C-8), 128.1 (C-
9), 128.0 (C-1ꢀ), 123.3 (C-2), 121.5 (C-6ꢀ), 120.4 (C-8ꢀ), 112.0 (C-5ꢀ), 111.8
(C-7), 111.5 (C-6), 111.0 (C-3), 110.2 (C-2ꢀ), 102.4 (C-4), 55.7 (3ꢀ-OMe),
55.6 (4ꢀ-OMe), 39.4 (C-11), 25.6 (C-10).
Brown oil. Yield 62.0%; IR ␯
max
NMR (400 MHz, DMSO- d6) ␦ 10.50 (1H, s, H-1), 8.00 (1H, t, 5.2 Hz,
-CONH), 7.14 (1H, d, 8.8 Hz, H-7), 7.05 (1H, s, H-2), 6.87 (1H, d, 1.6 Hz,
H-4), 6.62 (1H, dd, 8.8 Hz, 1.6 Hz, H-6), 3.30 (2H, dt, 5.2 Hz, 7.6 Hz, H-11),
2.73 (2H, t, 7.6 Hz, H-10). 2.44 (2H, t, 6.8 Hz, H-2ꢀꢀ), 2.35 (2H, t, 6.8 Hz,
H-3ꢀꢀ). 13C NMR (100 MHz, DMSO- d6) ␦ 174.7 (C-4ꢀꢀ), 172.8 (C-1ꢀꢀ),
150.4 (C-5), 131.0 (C-8), 128.1 (C-9), 123.2 (C-2), 111.8 (C-7), 111.4 (C-
6), 111.1 (C-3), 102.4 (C-4), 39.7 (C-11), 30.9 (C-2ꢀꢀ), 30.1 (C-3ꢀꢀ), 25.5
(C-10).
3.5. DPPH radical scavenging activity
3.2.7. N-Sinapoyl serotonin (8)
The scavenging activity of cinnamic acid derivatives for the DPPH radi-
cal was monitored according to the method of Gaspar et al. (2009). An
amount of 500 ␮l of a 0.5 mM methanolic DPPH solution was mixed in a
cuvette with 500 ␮l of cinnamic acid derivatives at different concentration
levels. These cuvettes were shaken vigorously. The cuvettes were allowed
to stand at 27 ◦C for 30 min, the absorbance was measured at 517 nm using a
U-1500 spectrophotometer. The percentage of radical scavenging activity
Paleyellowsolid:Yield 76.0%;mp. 169–172 ◦C. IR ␯max (KBr): 3308, 1650,
1604, 1512, 800 cm−1 1H NMR (400 MHz, DMSO- d6) ␦ 10.48 (1H, s,
.
H-1), 8.77 (1H, s, 4ꢀ-OH), 8.60 (1H, s, 5-OH), 8.03 (1H, t, 4.7 Hz, -CONH),
7.33 (1H, d, 15.6 Hz, H-7ꢀ), 7.12 (1H, d, 8.4 Hz, H-7), 7.06 (2H, s, H-2ꢀ and
H-6ꢀ), 7.05 (1H, s, H-2), 6.84 (1H, d, 2.4 Hz, H-4), 6.59 (1H, dd, 8.4 Hz,
2.4 Hz, H-6), 6.50 (1H, d, 15.6 Hz, H-8ꢀ), 3.78 (6H, s, 3ꢀ-OMe and 5ꢀ-OMe),
304
Pharmazie 66 (2011)

ORIGINAL ARTICLES
was calculated using the equation:
Choi SH, Hur JM, Yang EJ, Jun M, Park HJ, Lee KB, Moon E, Song KS
(2008) ␤-Secretase (BACE1) inhibitors from Perilla frutescens var. acuta.
Arch Pharm Res 31: 183–187.
Radical scavenging activity (%)
Marumoto S, Miyazawa M (2010) ␤-Secretase inhibitory effects of fura-
nocoumarins from the root of Angelica dahurica. Phytother Res 24:
510–513.
Sakamura S, Terayama Y, Kawakatsu S, Ichihara A, Saito H (1978) Con-
jugated serotonins related to cathartic activity in safflower (Carthamus
tinctorius L.) seeds. Agric Biol Chem 42: 1805–1806.
= (Control OD − Sample OD/Control OD) × 100
All tests were performed in triplicate. BHT as used as a reference standard
for the investigation of radical scavenging activity.
Sato H, Kawagishi H, Nishimura T, Yoneyama S, Yoshimoto Y, Sakamura S
(1985) Serotobenine, a novel phenolic amide from safflower (Carthamus
tinctorius L.) seeds. Agric Biol Chem 49: 2969–2974.
Zhang HL, Nagatsu A, Watanabe T, Sakakibara J, Okuyama H (1997)
Antioxidative compounds isolated from safflower (Carthamus tinctorius
L.) oil cake. Chem Pharm Bull 45: 1910–1914.
Roh JS, Han JY, Kim JH, Hwong JK (2004) Inhibitory effects of active
compounds isolated from safflower (Carthamus tinctorius L.) seeds for
melanogenesis. Biol Pharm Bull 27: 1976–1978.
Nagatsu A, Zhang H L, Mizukami H, Okuyama H, Sakakibara J, Tokuda
H, Nishino H (2000) Tyrosinase inhibitory and anti-tumor promoting
activities of compunds isolated from safflower (Carthamus tinctorius L.)
and cotton (Gossypium hirsutum L.) oil cakes. Nat Prod Lett 14: 153–158.
Takii T, Hayashi M, Hiroma H, Chiba T, Kawashima S, Zhang H
L, Nagatsu A, Sakakibara J, Onozaki K (1999). Serotonin deriva-
tive, N-(p-coumaroyl) serotonin, isolated from safflower (Carthamus
tinctorius L.) oil cake augments the proliferation of normal human
and mouse fibroblasts in synergy with basic fibroblasts growth fac-
tor (bFGF) or epidermal growth factor (EGF). J Biochem 125:
910–915.
3.6. β-Secretase inhibitory activity
BACE 1 (␤-sectretase) assay was carried out using the BACE 1 Assay Kit
(Invitrogen, USA) according to the manufactures’s instruction. Briefly, a
mixture of 10 ␮l of assay buffer (50 mM sodium acetate, pH 4.5), 10 ␮l of
BACE 1 (recombinant human BACE 1 1.0 U/ml), 10 ␮l of substrate (750 nM
Rh-EVNL-DAEFK-Quencherin50 mMammoniumbicarbonate), and10 ␮l
of sample dissolved in 30% DMSO was incubated for 60 min at room tem-
perature in the dark. The mixture was irradiated at 550 nm and the emission
intensity at 590 nm was recorded. The inhibition ratio was obtained by the
following equation:
Inhibition (%) = [1 − {(S − S0)/(C − C0)}] × 100
where C was the fluorescence of the control (enzyme, buffer and substrate)
after 60 min of incubation, C0 was the fluorescence of the control at zero
time, S was the fluorescence of the tested samples (enzyme, sample solution
and substrate) after incubation, and S0 was the fluorescence of the tested
samples at zero time. To allow for the quenching effect of the samples, the
sample solution was added to the reaction mixture C, and any reduction in
fluorescence by the sample was then investigated. All data are the mean of
three experiments.
Park JB (2008) Serotomide and safflomide modulate forskolin-stimulated
cAMP formation via 5-HT1 receptor. Phytomedicine 15: 1093–1098.
Yamazaki Y, Kawano Y, Yamanaka A, Maruyama S (2009) N-[(Dihy-
droxyphennyl)acyl]serotonins as potent inhibitors of tyrosinase from
mouse and human malanoma cells. Bioorg Med Chem Lett 19: 4178–
4182.
Kang K, Park S, Kim Y S, Lee S, Back K (2009) Biosynthesis and biotech-
nological production of serotonin derivatives. Appl Microbiol Biotechnol
83: 27–34.
Koyama N, Kuribayashi K, Seki T and Kobayashi K FuruhataY, Suzuki K,
Arisaka H, Nakano T, Amino Y and Ishii K (2006) Serotonin derivatives,
major safflower (Carthamus tinctorius L.) seed antioxidants, inhibit low-
density lipoprotein (LDL) oxidation and atherosclerosis in apolipoprotein
E-deficient Mice. J Agric Food Chem 54: 4970–4976.
Somei M, Yamada F, Kurauchi T, Nagahama Y, Hasegawa M, Yamada K,
Teranishi S, Sato H, Kaneko C (2001) The chemistry of indole.CIII.
Simple syntheses of serotonin, N-methylserotonin, bufotenine, 5-
methoxy-N- methyltryptamine, bufobutanoic acid, N-(indole-3-yl)
methy-5- methoxy-N-methyltryptamine, and lespedamine based on1-
hydroxyindole chemistry. Chem Pharm Bull 49: 87–96.
References
Prasad KN, Hovland AP, Cole WC, Prasad KC, Nahreini P, Prasad EJ,
Andreatta CP (2000) Multiple antioxidant in the prevention and treatment
ofAlzheimerdisease:analysisofbiologicrationale. ClinNeuropharmacol
23: 2–13.
Prasad KN, Cole WC, Prasad KC (2002) Risk factors for Alzheimer’s dis-
ease: Role of multiole antioxidants, non-steroidal anti-inflammatory and
cholinergic agents alone or in combination in prevention and treatment.
J Amer Coll Nutr 21: 506–522.
Dorrel S (2000) Untangling Alzheimer’s disease with beta-secretase
inhibitors. Drug Discov Today 5: 316–331.
Stachel SJ, Coburn CA, Steele TG, Jones KG, Loutzenhiser EF, Gregro AR,
Rajapakse HA, Lai MT, Crouthamel MC, Xu M, Tugusgeva K, Lineberger
JE, Pietrak BL, Espeseth AS, Shi XP, Chen-Dodson E, Holloway MK,
Munshi S, Simon AJ, Kuo L, Vacca JP (2004) Structure-based design
of potent and selective cellpermeable inhibitors of human ␤-secretase
(BACE-1). J Med Chem Lett 47: 6447–6450.
Parr AJ, Bolwell BP (2000) Phenols in the plant and in man. The potential
for possible nutritional enhancement of the diet by modifying the phenols
content or profile. J Sci Food Agri 80: 985–1012.
Gaspar A, Garrido E M, Esterves M, Quezada E, Milhazes N, Garrido J,
Borges F (2009) New insights into the antioxidant activity of hydroxycin-
namic acids: Synthesis and physicochemical characterization of novel
hagenated derivatives. Eur J Med Chem 44: 2092–2099.
JeonSY, BaeKH, SeongYH, SongKS(2003)GreenteacatechinsasaBACE
1 (beta-secretase) inhibitor. Bioorg Med Chem Lett 13: 3905–3908.
Pharmazie 66 (2011)
305