Synthesis and structure-activity relationships of serotonin derivatives effect on α-glucosidase inhibition

Article abstract of DOI:10.1007/s00044-011-9699-9

The α-glucosidase inhibitory activities of serotonin derivatives were evaluated. Two serotonin derivatives, N-p-coumaroyl serotonin (2) and N-caffeoyl serotonin (4) exhibited most potent inhibition on α-glucosidase, whereas, cinnamic acid derivatives were

Full text of DOI:10.1007/s00044-011-9699-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1762–1770
DOI 10.1007/s00044-011-9699-9
ORIGINAL RESEARCH
Synthesis and structure–activity relationships of serotonin
derivatives effect on a-glucosidase inhibition
•
Toshiyuki Takahashi Mitsuo Miyazawa
Received: 30 December 2010 / Accepted: 9 June 2011 / Published online: 18 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract The a-glucosidase inhibitory activities of sero-
tonin derivatives were evaluated. Two serotonin derivatives,
N-p-coumaroyl serotonin (2) and N-caffeoyl serotonin (4)
exhibited most potent inhibition on a-glucosidase, whereas,
cinnamic acid derivatives were less efficient. Furthermore,
we analyzed their structural importance for a-glucosidase
inhibition. The linkage of cinnamic acid moiety and sero-
tonin moiety and the olefin in cinnamic acid moiety of
serotonin derivatives were crucial for a-glucosidase inhibi-
tion. This is the first report on structure–activity relation-
ships (SAR) for the a-glucosidase inhibitory activity of
serotonin derivatives.
complications. Several studies on the treatment of type 2
diabetes suggest that improved glycemic control reduces
microvascular risk (Vijan et al., 1997; UKPDS group,
1998a, b; Gaster and Hirsch, 1998). There are reports of
established a-glucosidase inhibitors such as acarbose
(Schmidit et al., 1977) and voglibose (Matsuo et al., 1992)
from microorganisms, nojirimycin, and 1-deoxynojirimycin
(Asano et al., 1995; Chen et al., 1995) from plants. In recent,
many researchers have evaluated a-glucosidase inhibitors
from natural sources (Kim et al., 2008; Chougale et al.,
2009; Sim et al., 2010; Choi et al., 2010; Wang et al., 2010).
Glucosidase inhibitors have been very interesting biological
activities (Humphries et al., 1986; Gruters et al., 1987), thus,
there might be value in searching for glucosidase inhibitors
derived from a food in the view of safety.
Keywords Serotonin derivatives ꢀ
a-Glucosidase inhibitor ꢀ Diabetes ꢀ
Structure–activity relationship
Safflower (Carthamus tinctorius. L) is one of the
important medicinal plants in Asia country (Bae et al.,
2002). Especially, safflower seeds contained lignans (Palter
et al., 1972a), phytosterols (Palter et al., 1972b), and poly-
phenols (Zhang et al., 1997). Among these compounds,
serotonin derivatives, such as, N-p-coumaroyl serotonin and
N-feruloyl serotonin were identified as the major unique
phenolic compound of safflower seeds. These serotonin
derivatives have a number of biological effects, such as,
antioxidative activity (Zhang et al., 1997), anti-tyrosinase
activity, melanin-production inhibitory activity (Roh et al.,
2004), anti-tumor activity (Nagatsu et al., 2000), fibroblasts
growth promoting activity (Takii et al., 1999). Conse-
quently, a broad range of biological activities of serotonin
derivatives have been reported. In this study, the inhibitory
effects of the synthetic serotonin analogues (Table 1) on
a-glucosidase were evaluated to investigate a new pharma-
cological activity of serotonin derivatives, to study the
structure–activity relationships and kinetics of inhibitory
activity.
Introduction
a-Glucosidase inhibitors are applicable to the treatment of
numerous diseases including diabetes mellitus type 2 (Floris
et al., 2005), cancer (Fernandes et al., 1991), and HIV
(Ozawa et al., 2001). In particular, diabetes mellitus is the
most serious, chronic metabolic disorder and is character-
ized by high blood glucose levels (Corry and Tuck, 2000;
Stratton et al., 2000). Type 2 diabetes mellitus is a group
of disorders characterized by hyperglycemia and associ-
ated with microvascular, macrovascular, and neuropathic
T. Takahashi ꢀ M. Miyazawa (&)
Department of Applied Chemistry, Faculty of Science
and Engineering, Kinki University, 3-4-1, Kowakae,
Higashiosaka-shi, Osaka, Japan
e-mail: miyazawa@kindai.ac.jp
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organic extract was washed successively with 5% citric acid
solution (3 9 20 ml), saturated sodium hydrogen carbonate
solution (3 9 30 ml), and brine (50 ml). The extract was
dried over Na2SO4 and concentrated to yield a solid (yield
64.9–96.1%).
Table 1 The structures of serotonin derivatives (1–8)
Compound
R1
R2
R3
N-Cinnamoyl serotonin (1)
Yield 95.0%; m.p., 92–95°C. IR mmax (KBr): 3292, 1654,
1607, 1531, 798 cm-1. H NMR (400 MHz, DMSO-d6) d
1
1
2
3
4
5
6
7
8
H
H
H
10.48 (1H, s, H-1), 8.50 (1H, s, 5-OH), 8.19 (1H, t,
J = 5.6 Hz, –NH), 7.55 (2H, d, J = 6.8 Hz, H-20, and
H-60), 7.42 (1H, d, J = 16.0 Hz, H-70), 7.38 (3H, m, H-30,
H-40 and H-50), 7.12 (1H, d, J = 8.0 Hz, H-7), 7.05 (1H, s,
H-2), 6.85 (1H, d, J = 2.4 Hz, H-4), 6.63 (1H, d,
J = 16.0 Hz, H-80), 6.56 (1H, dd, J = 8.0 Hz, 2.4 Hz,
H-6), 3.44 (2H, dt, J = 5.6 Hz, 6.0 Hz, H-11), 2.79 (2H, t,
J = 6.0 Hz, H-10). 13C NMR (100 MHz, DMSO-d6) d
164.8 (C-90), 150.2 (C-5), 138.4 (C-70), 135.0 (C-10), 130.8
(C-8), 129.3 (C-40), 128.9 (C-20 and C-60), 127.9 (C-9),
127.5 (C-30and C-50), 123.1 (C-2), 122.4 (C-80), 111.6
(C-7), 111.3 (C-6), 110.8 (C-3), 102.2 (C-4), 39.5 (C-11),
25.4 (C-10).
H
OH
OMe
OH
OH
OMe
OMe
OH
H
H
H
H
OH
OMe
OH
OMe
OMe
H
H
H
OMe
Experimental
Thin layer chromatography (TLC) was performed on pre-
coated plates (silica gel 60 F254, 0.25 mm, Merk, Darms-
tadt, Germany). Column chromatography was carried
out using 70–230 mesh silica gel (Kieselgel 60, Merk,
Germany). Melting points (m.p.) were measured on a MP-
5000D melting-point apparatus and are uncorrected. 1H
and 13C NMR data were all obtained on JEOL ECA-400
(400 MHz), JEOL ECA-500 (500 MHz), and JEOL ECA-
700 (700 MHz) spectrometers in DMSO with TMS as
internal standard. IR spectra were obtained with a Jasco
FT/IR-470 plus Fourier transform infrared spectrometer.
Alpha-glucosidase (EC 3.2.1.20) was purchased from
Oriental Yeast Co., Ltd (Tokyo, Japan). p-Nitrophenyl-a-D-
glucopyranoside was purchased from Wako Pure Chemis-
try (Osaka, Japan). All solvents were purchased from
Kanto Chemical (Tokyo, Japan).
N-p-Coumaroyl serotonin (2)
Yield 75.0%; m.p., 192–195°C. FAB-MS m/z 323
[M ? H]? IR mmax (KBr): 3318, 1586, 1512, 830 cm-1. 1H
NMR (500 MHz, DMSO-d6) d 10.48 (1H, d, J = 2.3 Hz,
H-1), 8.60 (1H, s, 5-OH), 8.06 (1H, t, J = 5.6 Hz, –NH),
7.38 (2H, d, J = 8.6 Hz, H-20 and H-60), 7.33 (1H, d,
J = 16.0 Hz, H-70), 7.12 (1H, d, J = 8.6 Hz, H-7), 7.05
(1H, d, J = 2.3 Hz, H-2), 6.85 (1H, d, J = 2.3 Hz, H-4),
6.79 (2H, d, J = 8.6 Hz, H-30 and H-50), 6.59 (1H, dd,
J = 8.6 Hz, 2.3 Hz, H-6), 6.41 (1H, d, J = 16.0 Hz, H-80),
3.42 (2H, dt, J = 5.6 Hz, 7.8 Hz, H-11), 2.78 (2H, t,
J = 7.8 Hz, H-10). 13C NMR (125 MHz, DMSO-d6) d
165.5 (C-90), 159.0 (C-40), 150.4 (C-5), 138.7 (C-70), 131.0
(C-8), 129.4 (C-20 and C-60), 128.1 (C-9), 126.2 (C-10),
123.3 (C-2), 119.1 (C-80), 115.9 (C-30 and C-50), 111.8
(C-7), 111.5 (C-6), 111.0 (C-3), 102.4 (C-4), 40.0 (C-11),
25.6 (C-10).
Synthesis of serotonin derivatives (1–3, 5–8)
Synthesis of serotonin derivatives (1–3, 5–8) were accom-
plished according to our previous study (Takahashi and
Miyazawa, 2011). First, triethylamine (230 ll, 1.65 mmol),
1-hydroxybenzotriazole (223 mg, 1.65 mmol), and EDC
(290 ll, 1.65 mmol) were added to a solution serotonin
hydrochloride (350 mg, 1.65 mmol) and cinnamic acid
derivatives (1.50 mmol) in dinethylformamide (1.5 ml) and
dichloromethane (10 ml). After the mixture was stirred
over night at room temperature under nitrogen, it was
concentrated in vacuo. The residue was treated with water
(15 ml) and extracted with ethyl acetate (3 9 10 ml). The
N-p-Methoxy cinnamoyl serotonin (3)
Yield 79.2%; m.p., 178–185°C. IR mmax (KBr): 3271, 1650,
1601, 1574, 1509, 829 cm-1. 1H NMR (400 MHz, DMSO-
d6) d 10.50 (1H, d, J = 2.3 Hz, H-1), 8.12 (1H, t,
J = 6.0 Hz, –NH), 7.51 (2H, d, J = 8.8 Hz, H-20 and
H-60), 7.39 (1H, d, J = 15.6 Hz, H-70), 7.13 (1H, d,
J = 8.8 Hz, H-7), 7.06 (1H, d, J = 2.3 Hz, H-2), 6.86 (1H,
d, J = 2.0 Hz, H-4), 6.76 (2H, d, J = 8.8 Hz, H-30 and
H-50), 6.66 (1H, dd, J = 8.8 Hz, 2.0 Hz, H-6), 6.50 (1H, d,
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J = 15.6 Hz, H-80), 3.78 (3H, s, 40-OMe), 3.44 (2H, dt,
J = 6.0 Hz, 7.8 Hz, H-11), 2.79 (2H, t, J = 7.8 Hz, H-10).
13C NMR (100 MHz, DMSO-d6) d 165.1 (C-90), 160.2
(C-40), 150.2 (C-5), 138.1 (C-70), 130.8 (C-8), 129.0 (C-20
and C-60), 127.9 (C-9), 127.5 (C-10), 123.1 (C-2), 119.9
(C-80), 114.4 (C-30 and C-50), 111.6 (C-7), 111.3 (C-6), 110.8
(C-3), 102.2 (C-4), 55.2 (4-OMe), 39.4 (C-11), 25.4 (C-10).
(1H, d, J = 15.6 Hz, H-70), 7.15 (1H, d, J = 1.4 Hz, H-20),
7.13 (1H, d, J = 8.7 Hz, H-7), 7.11 (1H, dd, J = 8.4 Hz,
1.4 Hz, H-60), 7.07 (1H, s, H-2), 6.98 (1H, d, J = 8.4 Hz,
H-50), 6.86 (1H, d, J = 2.2 Hz, H-4), 6.60 (1H, dd,
J = 8.7 Hz, 2.2 Hz, H-6), 6.53 (1H, d, 15.6 Hz, H-80), 3.80
(3H, s, 30-OMe), 3.78 (3H, s, 40-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) d 165.4 (C-90), 150.4 (C-5), 150.2
(C-40), 149.1 (C-30), 138.7 (C-70), 131.0 (C-8), 128.1 (C-9),
128.0 (C-10), 123.3 (C-2), 121.5 (C-60), 120.4 (C-80), 112.0
(C-50), 111.8 (C-7), 111.5 (C-6), 111.0 (C-3), 110.2 (C-20),
102.4 (C-4), 55.7 (30-OMe), 55.6 (40-OMe), 39.4 (C-11),
25.6 (C-10).
N-Feruloyl serotonin (5)
Yield 96.1%; m.p., 110–114°C. IR mmax (KBr): 3374, 1652,
1
1589, 1514, 844 cm-1. H NMR (500 MHz, DMSO-d6) d
10.49 (1H, d, J = 2.0 Hz, H-1), 9.42 (1H, s, 40-OH), 8.61
(1H, s, 5-OH), 8.05 (1H, t, J = 5.8 Hz, –NH), 7.34 (1H, d,
J = 15.6 Hz, H-70), 7.13 (1H, d, J = 8.6 Hz, H-7), 7.12
(1H, d, J = 2.0 Hz, H-20), 7.06 (1H, d, J = 2.0 Hz, H-2),
6.99 (1H, dd, J = 8.1 Hz, 2.0 Hz, H-60), 6.86 (1H, d,
J = 2.3 Hz, H-4), 6.79 (1H, d, J = 8.1 Hz, H-50), 6.60
(1H, dd, J = 8.6 Hz, 2.3 Hz, H-6), 6.46 (1H, d,
J = 15.6 Hz, H-80), 3.81 (3H, s, 30-OMe), 3.43 (2H, dt,
J = 5.8 Hz, 7.5 Hz, H-11), 2.78 (2H, t, J = 7.5 Hz, H-10).
13C NMR (125 MHz, DMSO-d6) d 165.5 (C-90), 150.4
(C-5), 148.4 (C-40), 148.0 (C-30), 139.0 (C-70), 131.0 (C-8),
128.1 (C-9), 126.7 (C-10), 123.3 (C-2), 121.7 (C-60), 119.4
(C-80), 115.8 (C-50), 111.9 (C-7), 111.5 (C-6), 111.0 (C-3),
110.9 (C-20), 102.4 (C-4), 55.7 (30-OMe), 39.6 (C-11), 25.6
(C-10).
N-Sinapoyl serotonin (8)
Yield 76.0%; m.p., 169–172°C. IR mmax (KBr): 3308, 1650,
1
1604, 1512, 800 cm-1. H NMR (400 MHz, DMSO-d6) d
10.48 (1H, s, H-1), 8.77 (1H, s, 40-OH), 8.60 (1H, s, 5-OH),
8.03 (1H, t, J = 4.7 Hz, –NH), 7.33 (1H, d, J = 15.6 Hz,
H-70), 7.12 (1H, d, J = 8.4 Hz, H-7), 7.06 (2H, s, H-20 and
H-60), 7.05 (1H, s, H-2), 6.84 (1H, d, J = 2.4 Hz, H-4),
6.59 (1H, dd, J = 8.4 Hz, 2.4 Hz, H-6), 6.50 (1H, d,
J = 15.6 Hz, H-80), 3.78 (6H, s, 30-OMe and 50-OMe), 3.42
(2H, dt, J = 4.7 Hz, 7.2 Hz, H-11), 2.79 (2H, t,
J = 7.2 Hz, H-10). 13C NMR (100 MHz, DMSO-d6) d
165.3 (C-90), 150.2 (C-5), 148.0 (C-30 and C-50), 139.1
(C-70), 137.2 (C-40), 130.8 (C-8), 127.9 (C-9), 125.4 (C-10),
123.1 (C-2), 119.6 (C-80), 111.6 (C-7), 111.3 (C-6), 110.8
(C-3), 105.2 (C-20 and C-60), 102.2 (C-4), 55.9 (30-OMe
and 50-OMe), 39.4 (C-11), 25.4 (C-10).
N-Isoferuloyl serotonin (6)
Yield 77.0%; m.p., 99–105°C. IR mmax (KBr): 3390, 1652,
1
1584, 1509, 801 cm-1. H NMR (400 MHz, DMSO-d6) d
10.47 (1H, d, J = 2.0 Hz, H-1), 9.16 (1H, s, 30-OH), 8.59
(1H, s, 5-OH), 8.10 (1H, t, J = 6.0 Hz, –NH), 7.27 (1H, d,
J = 15.6 Hz, H-70), 7.10 (1H, d, J = 8.0 Hz, H-7), 7.04
(1H, d, J = 2.0 Hz, H-2), 6.97 (1H, d, J = 1.2 Hz, H-20),
6.95 (1H, dd, J = 8.4 Hz, 1.2 Hz, H-60), 6.92 (1H, d,
J = 8.4 Hz, H-50), 6.86 (1H, d, J = 1.6 Hz, H-4), 6.58
(1H, dd, J = 8.0 Hz, 1.6 Hz, H-6), 6.39 (1H, d,
J = 15.6 Hz, H-80), 3.78 (3H, s, 40-OMe), 3.41 (2H, dt,
J = 6.0 Hz, 7.2 Hz, H-11), 2.77 (2H, t, J = 7.2 Hz, H-10).
13C NMR (100 MHz, DMSO-d6) d 165.1 (C-90), 150.2
(C-5), 149.1 (C-40), 146.7 (C-30), 138.5 (C-70), 130.8 (C-8),
127.9 (C-9), 127.8 (C-10), 123.1 (C-2), 120.1 (C-60), 119.7
(C-80), 113.3 (C-50), 112.1 (C-7), 111.6 (C-6), 111.3 (C-3),
110.8 (C-20), 102.4 (C-4), 55.6 (40-OMe), 39.4 (C-11), 25.4
(C-10).
Synthesis of N-caffeoylserotonin (4)
First, to a solution of 3,4-dihydroxybenzaldehyde (297 mg,
2.15 mmol) in anhydr. 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 9 3).
The organic extract was washed successively with water
(3 9 10 ml) and brine (50 ml). The extract was dried over
Na2SO4 and concentrated to obtained 3,4-diallyloxybenz-
aldehyde (459.3 mg, yield 98.0%). Secondly, 3,4-diallyl-
oxybenzaldehyde and malonic acid were dissolved in
anhydr. 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 2 N 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-diallyloxy cinnamic acid (453.7 mg, yield 82.6%),
to the following, triethylamine (230 ll, 1.65 mmol),
1-hydroxybenzotriazole (223 mg, 1.65 mmol), and EDC
N-3,4-Dimethoxy cinnamoyl serotonin (7)
Yield 82.0%; m.p., 103–105°C. IR mmax (KBr): 3369, 1654,
1
1595, 1542, 802 cm-1. H NMR (700 MHz, DMSO-d6) d
10.49 (1H, s, H-1), 8.07 (1H, t, J = 5.6 Hz, –NH), 7.37
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Med Chem Res (2012) 21:1762–1770
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(290 ll, 1.65 mmol) were added to a solution serotonin
hydrochloride (350 mg, 1.65 mmol) and 3,4-diallyloxy
cinnamic acid (1.50 mmol) in dimethylformamide (1.5 ml)
and dichloromethane (10 ml). After the mixture was stirred
over night at room temperature under nitrogen, it was
concentrated in vacuo. The residue was treated with water
(15 ml) and extracted with ethyl acetate (3 9 10 ml). The
organic extract was washed successively with 5% citric
acid solution (3 9 20 ml), saturated sodium hydrogen
carbonate solution (3 9 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 anhydr. 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 temperature (monitored by TLC) and concentrated
in vacuo. The residue was treated with saturated NH4Cl
solution and extracted with ethyl acetate (3 9 10 ml). The
extract was dried 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 compound 4 (422.0 mg, yield 83.3%).
nitrogen, it was concentrated in vacuo. The residue was
treated with water (15 ml) and extracted with ethyl acetate
(3 9 10 ml). The organic extract was washed successively
with 5% citric acid solution (3 9 20 ml), saturated sodium
hydrogen carbonate solution (3 9 30 ml), and brine
(50 ml). The extract was dried over Na2SO4 and concen-
trated to yield a solid (yield 44.4–85.0%).
N-Dihydrocinnamoyl serotonin (1a)
Yield 44.4%; m.p., 48–50°C. IR mmax (KBr): 3331, 1632,
1
1586, 1531, 932 cm-1. H NMR (400 MHz, DMSO-d6) d
10.47 (1H, s, H-1), 7.91 (1H, t, J = 5.6 Hz, –NH), 7.29
(5H, m, H-20, H-30, H-40, H-50 and H-60), 7.12 (1H, d,
J = 8.0 Hz, H-7), 6.99 (1H, s, H-2), 6.83 (1H, d,
J = 2.4 Hz, H-4), 6.62 (1H, dd, J = 2.4 Hz, 8.0 Hz, H-6),
3.28 (2H, dt, J = 5.6 Hz, 7.2 Hz, H-11), 2.82 (2H, t,
J = 7.2 Hz, H-70), 2.68 (2H, t, J = 7.2 Hz, H-10), 2.37
(2H, t, J = 7.2 Hz, H-80). 13C NMR (100 MHz, DMSO-d6)
d 171.1 (C-90), 150.1 (C-5), 141.1 (C-10), 130.8 (C-8),
128.2 (C-20 and C-60), 128.1 (C-30 and C-50), 127.9 (C-9),
125.8 (C-40), 123.0 (C-2), 111.6 (C-7), 111.2 (C-6), 110.9
(C-3), 102.2 (C-4), 39.5 (C-11), 37.1 (C-80), 31.1 (C-70),
25.4 (C-10).
N-Caffeoyl serotonin (4)
Total yield 64.9%; m.p., 98–102°C. IR mmax (KBr): 3409,
1647, 1575, 1538, 811 cm-1. 1H NMR (400 MHz, DMSO-
d6) d 10.51 (1H, s, H-1), 8.14 (1H, t, J = 5.2 Hz, –NH),
7.27 (1H, d, J = 15.6 Hz, H-70), 7.14 (1H, d, J = 8.0 Hz,
H-7), 7.07 (1H, s, H-2), 6.98 (1H, d, J = 2.0 Hz, H-20),
6.87 (1H, d, J = 2.0 Hz, H-4), 6.85 (1H, dd, J = 2.0 Hz,
8.0 Hz, H-60), 6.77 (1H, d, J = 8.0 Hz, H-50), 6.61 (1H, dd,
J = 8.0 Hz, 2.0 Hz, H-6), 6.37 (1H, d, J = 15.6 Hz, H-80),
3.43 (2H, dt, J = 5.2 Hz, 7.6 Hz, H-11), 2.79 (2H, t,
J = 7.6 Hz, H-10). 13C NMR (100 MHz, DMSO-d6) d
165.4 (C-90), 150.2 (C-5), 147.3 (C-40), 145.6 (C-30), 139.0
(C-70), 130.8 (C-8), 127.9 (C-9), 126.5 (C-10), 123.1 (C-2),
120.4 (C-60), 118.8 (C-80), 115.8 (C-50), 113.9 (C-20), 111.7
(C-7), 111.3 (C-6), 110.9 (C-3), 102.3 (C-4), 39.5 (C-11),
25.5 (C-10).
N-Dihydro-p-coumaroylserotonin (2a)
Yield 83.7%; m.p., 60–63°C. IR mmax (KBr): 3414, 1631,
1
1513, 823 cm-1. H NMR (400 MHz, DMSO-d6) d 10.48
(1H, s, H-1), 7.90 (1H, t, J = 5.2 Hz, –NH), 7.15 (1H, d,
J = 8.4 Hz, H-7), 7.00 (1H, s, H-2), 6.99 (2H, d,
J = 8.4 Hz, H-20 and H-60), 6.86 (1H, d, J = 2.4 Hz, H-4),
6.69 (2H, d, J = 8.4 Hz, H-30 and H-50), 6.62 (1H, dd,
J = 8.4 Hz, 2.4 Hz, H-6), 3.29 (2H, dt, J = 5.2 Hz,
7.2 Hz, H-11), 2.73 (2H, t, J = 7.2 Hz, H-10), 2.71 (2H, t,
J = 7.2 Hz, H-70), 2.33 (2H, t, J = 7.2 Hz, H-80). 13C
NMR (100 MHz, DMSO-d6) d 171.4 (C-90), 155.4 (C-40),
150.2 (C-5), 131.5 (C-10), 130.8 (C-8), 129.0 (C-20 and
C-60), 127.9 (C-9), 123.0 (C-2), 115.0 (C-30 and C-50),
111.6 (C-7), 111.3 (C-6), 110.9 (C-3), 102.2 (C-4), 39.4
(C-11), 37.7 (C-80), 30.4 (C-70), 25.4 (C-10).
Synthesis of dihydroserotonin derivatives (1a, 2a, 5a, 8a)
N-Dihydroferuloyl serotonin (5a)
First, synthesis of dihydrocinnamic acid derivatives was
accomplished according to previous report (Takahashi
and Miyazawa, 2010). Secondly, triethylamine (230 ll,
1.65 mmol), 1-hydroxybenzotriazole (223 mg, 1.65 mmol),
and EDC (290 ll, 1.65 mmol) were added to a solution
serotonin hydrochloride (350 mg, 1.65 mmol) and dihy-
drocinnamic acid derivatives (1.50 mmol) in dinethylfor-
mamide (1.5 ml) and dichloromethane (10 ml). After the
mixture was stirred over night at room temperature under
Yield 85.0%; m.p., 62–65°C. IR mmax (KBr): 3385, 1635,
1
1515, 800 cm-1. H NMR (400 MHz, DMSO-d6) d 10.47
(1H, d, J = 2.4 Hz, H-1), 7.90 (1H, t, J = 6.4 Hz, –NH),
7.12 (1H, d, J = 8.0 Hz, H-7), 7.01 (1H, d, J = 2.4 Hz,
H-2), 6.82 (1H, d, J = 2.4 Hz, H-4), 6.76 (1H, d,
J = 2.4 Hz, H-20), 6.66 (1H, dd, J = 8.0 Hz, 2.4 Hz, H-6),
6.59 (1H, dd, J = 8.0 Hz, 2.4 Hz, H-60), 6.58 (1H, d,
J = 8.0 Hz, H-50), 3.73 (3H, s, 30-OMe), 3.28 (2H, dt,
123

1766
Med Chem Res (2012) 21:1762–1770
a-Glucosidase inhibitory activity
J = 6.4 Hz, 7.2 Hz, H-11), 2.71 (2H, t, J = 7.2 Hz, H-10),
2.69 (2H, t, J = 7.2 Hz, H-70), 2.33 (2H, t, J = 7.2 Hz,
13
H-80). C NMR (100 MHz, DMSO-d6) d 171.4 (C-90),
a-Glucosidase inhibitory activity was determined using the
modified version of the method according to Li et al.
(2007). a-Glucosidase (25 ll, 0.2 U/ml), 25 ll of various
concentrations of samples, and 175 ll of 50 mM sodium
phosphate buffer (pH 7.0) were mixed at room temperature
for 10 min. The reaction was started by the addition of
25 ll of 2.5 mM p-nitrophenyl-a-D-glucopyranoside. The
reaction mixture was incubated for 10 min at 37°C. The
activities of glucosidase were detected in 96-well plate, and
the absorbance was determined at 405 nm (for p-nitro-
phenol) in a microplate reader (Corona Electric Co., Ltd).
Control sample contained 25 ll DMSO in place of test
samples. Percentage of enzyme inhibition was calculated as
(1 - B/A)C 9 100, where A represents absorbance of
control without test samples, and B represents absorbance
in presence of test samples. All the tests were run in
duplicate. Acarbose and 1-deoxynojirimycin were used as
the positive control in this study. The fifty percent inhibi-
tory concentration (IC50) values were expressed as
mean SE (n = 3).
150.2 (C-5), 147.3 (C-40), 144.5 (C-30), 132.2 (C-10), 130.8
(C-8), 127.9 (C-9), 123.0 (C-2), 120.2 (C-60), 115.3 (C-50),
112.4 (C-7), 111.6 (C-6), 111.2 (C-20), 110.9 (C-3), 102.2
(C-4), 55.5 (30-OMe), 39.4 (C-11), 37.6 (C-80), 30.8 (C-70),
25.4 (C-10).
N-Dihydrosinapoyl serotonin (8a)
Yield 71.1%; m.p., 155–157°C. IR mmax (KBr): 3386, 1646,
1
1558, 1518, 801 cm-1. H NMR (400 MHz, DMSO-d6) d
10.46 (1H, s, H-1), 7.90 (1H, t, J = 6.8 Hz, –NH), 7.11
(1H, d, J = 8.4 Hz, H-7), 7.01 (1H, s, H-2), 6.83 (1H, d,
J = 1.6 Hz, H-4), 6.59 (1H, dd, J = 8.4 Hz, 1.6 Hz, H-6),
6.46 (2H, s, H-20 and H-60), 3.72 (6H, s, 30-OMe and 50-
OMe), 3.28 (2H, dt, J = 6.8 Hz, 7.2 Hz, H-11), 2.72 (2H,
t, J = 7.6 Hz, H-70), 2.70 (2H, t, J = 7.2 Hz, H-10), 2.34
(2H, t, J = 7.6 Hz, H-80). 13C NMR (100 MHz, DMSO-d6)
d 171.3 (C-90), 150.1 (C-5), 147.8 (C-30 and C-50), 133.6
(C-10), 131.4 (C-40), 130.8 (C-8), 127.9 (C-9), 123.0 (C-2),
111.6 (C-7), 111.2 (C-6), 110.9 (C-3), 105.6 (C-20 and
C-60), 102.2 (C-4), 55.9 (30-OMe and 50-OMe), 39.4 (C-11),
37.5 (C-80), 31.3 (C-70), 25.4 (C-10).
Results and discussion
The a-glucosidase inhibitory activities of serotonin deriv-
atives (1–8) were investigated and compared with that of
acarbose and 1-deoxynojirimycin, a clinically used a-glu-
cosidase inhibitors, were employed as a reference drugs.
These results were shown in Fig. 1. All serotonin deriva-
tives (1–8) demonstrated a significant inhibition on a-glu-
cosidase in a dose dependent manner. In particular,
compound 2 and 4 showed excellent inhibitions on yeast
a-glucosidase a with extremely high potency, and their
Synthesis of N-dihydrocaffeoyl serotonin (4a)
N-Caffeoylserotonin (1.1 mmol) was dissolved in 8 ml
methanol, palladium on carbon (10%, wet, 200 mg) was
added and the mixture was stirred under H2 at atmospheric
pressure at room temperature for 2 h. The catalyst was
filtered through a bed of CeliteÒ and solvent was evapo-
rated under reduced pressure to yield 95.0%.
N-Dihydrocaffeoylserotonin (4a)
Yield 95.0%; m.p., 78–80°C. IR mmax (KBr): 3366, 3184,
1615, 1556, 1488, 799 cm-1. 1H NMR (400 MHz, DMSO-
d6) d 10.46 (1H, s, H-1), 7.88 (1H, t, J = 5.2 Hz, –NH),
7.10 (1H, d, J = 8.0 Hz, H-7), 7.00 (1H, s, H-2), 6.82 (1H,
d, J = 2.0 Hz, H-4), 6.78 (1H, d, J = 7.6 Hz, H-50), 6.57
(1H, dd, J = 8.0 Hz, 2.0 Hz, H-6), 6.25 (1H, d,
J = 2.0 Hz, H-20), 6.11 (1H, dd, J = 2.0 Hz, 7.6 Hz,
H-60), 3.26 (2H, dt, J = 5.2 Hz, 7.0 Hz, H-11), 2.68 (2H, t,
J = 7.0 Hz, H-10), 2.62 (2H, t, J = 7.2 Hz, H-70), 2.26
(2H, t, J = 7.2 Hz, H-80). 13C NMR (100 MHz, DMSO-d6)
d 171.9 (C-90), 156.4 (C-40), 155.7 (C-30), 150.1 (C-5),
132.0 (C-10), 130.8 (C-8), 127.9 (C-9), 123.0 (C-2), 118.7
(C-60), 115.6 (C-50), 115.4 (C-20), 111.6 (C-7), 111.2 (C-6),
110.9 (C-3), 102.2 (C-4), 39.4 (C-11), 36.1 (C-80), 25.4
(C-10), 25.2 (C-70).
Fig. 1 Dose-dependent inhibitory effects of serotonin derivatives
(1–8). Acarbose and 1-deoxynojirimycin were used a positive control.
The result represents the means of triplicate experiments
123

Med Chem Res (2012) 21:1762–1770
1767
well as cinnamic acid moiety and serotonin moiety are
critical factor of a-glucosidase inhibitory activity. These
results accorded with previous reports that a-glucosidase
was inhibited to a great extent when the compound con-
tained a fused-aromatic ring and a heteroaromatic compo-
nent (Liang et al., 2006) and trans-cinnamic derivatives
showed a-glucosidase inhibitory activity (Adisakwattana
et al., 2004). Furthermore, we evaluated the inhibition of
dihydroserotonin derivatives to clarify the effects of olefin in
cinnamic acid moiety of serotonin derivatives.
Table 2 Inhibitory effects of serotonin derivatives
Compound
ICa50 (lM)
Compound
ICa50 (lM)
1
2
3
4
5
6
7
8
80.8 0.8
47.2 0.4
[300
1a
2a
4a
5a
8a
[300
[300
[300
[300
[300
48.2 0.6
99.8 2.2
[300
[300
66.2 1.3
Serotonin
[300
N-Dihydrocinnamoyl serotonin (1a), N-dihydro-p-cou-
maroyl serotonin (2a), N-dihydrocaffeoyl serotonin (4a),
N-dihydroferuloyserotonin (5a), and N-dihydro sinapoyl
serotonin (8a) decreased the inhibitory activity very much
(Table 2). The result indicated that the olefin in serotonin
derivatives is essential for a-glucosidase inhibition. From
the structural-activity point of view, compound 2, which
have OH group at 40 position exhibited an increase in a-
glucosidase inhibitory activity relative to compound 1. In
the contrast, compound 3, which have OMe group at 40
position decreased the a-glucosidase inhibitory activity. In
the case of compound 2, 30 position replacement with OH
group (compound 4) is similar to that of compound 2,
however, 30 position replacement with OMe group (com-
pound 5) decreases the inhibitory activity. Furthermore, the
introduction of another methoxy group in an ortho-position
to a hydroxy group (compound 8) led to increase inhibitory
activity relative to compound 5. On the other hand, in the
case of compound 3, when the meta-methoxy function of
compound 3 was substituted by a hydroxy group (com-
pound 6) or a methoxy group (compound 7), these com-
pounds exhibited low inhibitory activity. The inhibitory
effect of each compound on a-glucosidase activity is
shown in Table 2.
Cinnamic acid [300
p-Coumaric acid
[300
[300
Caffeic acid
Sinapic acid
Acarboseb
a
[300
145.3 1.2
Ferulic acid
907.5 2.2 1-Deoxynojirimycinb 278.0 1.7
Data represents means SD of triplicate samples
b
This compound was used as a positive control
50% inhibition of enzyme activity (IC50) values were cal-
culated as 47.2 lM (2) and 48.2 lM (4), respectively, which
were much lower than that of reference drugs, acarbose
(907.5 lM) and 1-deoxynojirimycin (278.0 lM). Other
active compounds, compound 1, 5, and 8, also exhibited a
potent inhibition with IC50 values of 80.8 lM (1), 99.8 lM
(5), and 66.2 lM (8), which were much more potent to
that of 1-deoxynojirimycin, respectively. Furthermore, we
evaluated the inhibitory activities of cinnamic acid deriva-
tives and serotonin to clarify whether inhibitory activities of
compound 1, 2, 4, 5, and 8 are due to cinnamic acid moiety or
serotonin moiety. Cinnamic acid (inhibition: 4.5 2.1% at
300 lM), p-coumaric acid (inhibition: 4.9 2.5% at
300 lM), caffeic acid (inhibition: 10.2 2.1 at 300 lM),
and serotonin (inhibition: 25.6 3.1% at 300 lM) exhib-
ited low inhibitory activity, on the other hand, sinapic
acid showed potent a-glucosidase inhibitory activity
(IC50 = 145.3 lM). However, each cinnamic acid deriva-
tives exhibited a relatively poor inhibitory activity as com-
pared with these serotonin derivatives. Therefore, we
considered the linkage of cinnamic acid and serotonin as
Inhibitory mechanisms of compound 1, 2, 4, 5, and 8 on
a-glucosidase were analyzed by a Lineweaver–Burk plot.
Inhibitory mechanism of compound 1 was competitive
type. In the presence of compound 1, the kinetics of the
enzyme, by double-reciprocal plots are shown in Fig. 2.
The plot of 1/v versus 1/[S] gave a family of straight lines
Fig. 2 a Lineweaver–Burk
plots for the inhibition of the
hydrolysis activity of
a-glucosidase by compound 1
(0, 80, 300 lM). b Dixon plots
for the inhibition of compound 1
(0, 80, 300 lM)
123

1768
Med Chem Res (2012) 21:1762–1770
Fig. 3 Lineweaver–Burk plots
for the inhibition of the
hydrolysis activity of a-
glucosidase by compound 2
(a) and 4 (b)
Fig. 4 Lineweaver–Burk plots
for the inhibition of the
hydrolysis activity of a-
glucosidase by compound 5
(a) and 8 (b)
with different slopes but they intersected one another in the
Y-axis. The values of Vmax remained the same and the
values of Km increased with increasing concentrations of
the inhibitor. The result showed that compound 1 could
only bind with free enzyme. The Ki (inhibition constant)
value of the compound 1 was determined to be 133.2 lM
by using Dixon plot (Fig. 2b). The inhibitory mechanisms
of compound 2 and 4 were uncompetitive type. Figure 3a
and b shows double reciprocal plots of enzyme inhibited by
compound 2 and 4, respectively. These results show that
the plot of 1/v versus 1/[S] give a family of parallel straight
lines with the same slopes. Accompanying the increase of
the inhibitor concentration the values of both Km and Vm
increased, but the ratio of Km/Vm remained unchanged.
The slopes were independent of the concentration of
compound 2 or compound 4 which indicated that com-
pound 2 and 4 were an uncompetitive inhibitor of the
enzyme. The Ki values of compound 2 and 4 were 81.6 and
123

Med Chem Res (2012) 21:1762–1770
1769
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In conclusion, we investigated the a-glucosidase inhibition
of phenylpropanoid amides of serotonin, to our knowledge;
there is no report on the inhibition of the other serotonins.
In this study, we reported the correlation between a-glu-
cosidase inhibition and structures of serotonin derivatives
for the first time. Compound 2 (IC50 = 47.2 lM) and
compound 4 (IC50 = 47.2 lM) showed a most potent
a-glucosidase. From the structural-activity point of view,
linkage of cinnamic acid and serotonin as well as cinnamic
acid moiety and serotonin moiety are critical factor of
a-glucosidase inhibitory activity, furthermore, the olefin in
serotonin derivatives is essential for inhibition. These
serotonin derivatives exhibited a potent inhibition on yeast
a-glucosidase in vitro and could be employed as a lead for
the design of new potential a-glucosidase inhibitors. In
addition further studies on the inhibitory activity of sero-
tonin derivatives against mammalian intestinal a-glucosi-
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