A simple synthesis of trans-3,4,5-trimethoxycinnamamides and evaluation of their biologic activity

Article abstract of DOI:10.1007/s00044-012-0415-1

A simple synthesis and biologic evaluation of trans-3,4,5- trimethoxycinnamamides 10a-e and 11 as novel antinarcotic agents is described. The synthetic key strategies involve condensation reaction and coupling reaction to generate trans-3,4,5-trimethoxycinnamamides 10a-e and 11. They were evaluated for free radical scavenging, inhibitory action for neurotoxicity in cultured neurons, and antinarcotic activity in mice. It was found that compounds 10a, 10d, and 10e displayed significant inhibitory action of the glutamate-induced neurotoxicity and 10a-e and 11 showed high antinarcotic activity in mice.

Full text of DOI:10.1007/s00044-012-0415-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4615–4621
DOI 10.1007/s00044-012-0415-1
ORIGINAL RESEARCH
A simple synthesis of trans-3,4,5-trimethoxycinnamamides
and evaluation of their biologic activity
•
Jae-Chul Jung Dongguk Min Heena Lim Sohyeon Moon
•
•
•
•
Mankil Jung Seikwan Oh
Received: 6 March 2012 / Accepted: 11 December 2012 / Published online: 17 January 2013
Ó Springer Science+Business Media New York 2013
Abstract A simple synthesis and biologic evaluation of
trans-3,4,5-trimethoxycinnamamides 10a–e and 11 as
novel antinarcotic agents is described. The synthetic key
strategies involve condensation reaction and coupling
reaction to generate trans-3,4,5-trimethoxycinnamamides
10a–e and 11. They were evaluated for free radical scav-
enging, inhibitory action for neurotoxicity in cultured
neurons, and antinarcotic activity in mice. It was found that
compounds 10a, 10d, and 10e displayed significant inhib-
itory action of the glutamate-induced neurotoxicity and
10a–e and 11 showed high antinarcotic activity in mice.
antioxidant (Carlotti et al., 2008), and antipyretic effect
(Sultana et al., 2005; Kurokawa et al., 1998). In particular,
trans-cinnamic acids and its derivatives are very effective to
modulate central nervous system (CNS) including attention
deficit hyperactivity disorder (ADHD), narcolepsy effects
(Nardi et al., 2008), and antidepressant-like effects were
related to sedative activity by suppressing norepinephrine
content in locus coeruleus (Kawashima et al., 2004) (Fig. 1).
3,4,5-Trimethoxycinnamic acid (TMCA) is a compo-
nent of the root of Polygala tenuifolia WILLLDENOW
(polygalaceae) and its extract was used as a sedative in
Japanese traditional Kampo medicine (Kawashima et al.,
2004). The Kawashima group studied the effect of TMCA
on stress-induced corticotrophin-releasing hormone (CRH)
release and found that TMCA exhibits sedative effects by
suppressing norepinephrine (NE) content in the locus
coeruleus (LC) (Kawashima et al., 2004). Hyperactivity of
LC neurons is an important component of opiate with-
drawal. Opiate withdrawal syndrome is mediated by an
excitatory amino acid (glutamate) input to the LC and
causes hyperactivity in the LC region (Akaoka and Aston-
Jones, 1993). To determine the development of morphine
dependence in mice or rats, naloxone (opioid receptor
antagonist) was injected and jumping behavior was mea-
sured (Jung et al., 2011). During naloxone-induced jump-
ing behavior, there was a further significant increase in the
extracellular levels of NE (Fuentealba et al., 2000). These
studies suggest that TMCA may be working at opioid
withdrawal by suppressing NE or blocking excitatory
amino acid input to the LC. Recently, Keung group (Tao
et al., 2005) demonstrated that aromatic-substituted cin-
namic acid and its structural analogs inhibit monoamine
oxidase A and exhibit antidepressant-like activity in mice.
The Geraci group (Consoli et al., 2006) described the
synthesis of hydroxycinnamic acid clustered by a calixarene
Keywords Trans-3,4,5-trimethoxycinnamamides ꢀ
Condensation reaction ꢀ Coupling reaction ꢀ
Antineurotoxicity ꢀ Free radical scavenging ꢀ
Antinarcotic activity
Introduction
Many methoxycinnamic acids and their derivatives have been
of interest for their antibacterial (Srivastava et al., 2007),
antiviral (King et al., 2009; Lee et al., 2007), anti-inflam-
matory (Chen et al., 2007), antitumor (Fong et al., 2008),
J.-C. Jung ꢀ H. Lim ꢀ S. Moon ꢀ S. Oh (&)
Department of Neuroscience and TIDRC, School of Medicine,
Ewha Womans University, Seoul 158-710, Korea
e-mail: skoh@ewha.ac.kr
J.-C. Jung
e-mail: jcjung10@yahoo.co.kr
D. Min ꢀ M. Jung
Department of Chemistry, Yonsei University,
Seoul 120-749, Korea
e-mail: mjung@yonsei.ac.kr
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Med Chem Res (2013) 22:4615–4621
OH
OH
O
O
O
R1
R2
MeO
HO
OMe
OH
OH
O
O
R3
COOH
5 Curcumin
1 Cinnamic acid; R1=R2=R3=H
2 p-Coumaric acid; R1=R2=OH, R3=H
3 Sinapic acid; R1=R3=OMe, R2=OH
OH
OH
OH
4 Chlorogenic acid
Fig. 1 Chemical structures of cinnamic acid (1), p-coumaric acid (2), sinapic acid (3), chlorogenic acid (4), and curcumin (5)
and evaluated its radical scavenging and antioxidant
activity using 1,1-diphenyl-2-picryl hydrazyl (DPPH)-
induced radicals and the 2,20-azobis(isobutyronitrile)
(AIBN)-induced linoleic acid peroxidation test. Priorities of
current research for the trans-3,4,5-methoxycinnamamides
are focused on antioxidant effect, inhibitory action for
the glutamate-induced neurotoxicity in neuronal cells, and
antinarcotic activity in mice. Previously, we reported that
trans-3,4,5-methoxyphenylacrylamides showed antinarcot-
ic activity in mice (Jung et al., 2010). Recent reports
described a simple synthesis and structural modification of
trans-3,4,5-trimethoxycinnamamides by Pathan group
(Pathan and Patil, 2008) and Nudelman group (Hedvati
et al., 2002), respectively. The Zhang group (Yang et al.,
2010) has developed to a new protocol for the synthesis of
trans-3,4,5-trimethoxycinnamamide analogs through silica
gel-mediated amide bond formation.
light. Flash chromatography was carried out on silica gel
60 [Scientific Adsorbents Incorporated (SAI), particle size
32–63 lM, pore size 60 A]. 1H NMR and 13C NMR
˚
spectra were recorded on a Bruker DPX 500 at 500 and
125 MHz, respectively. The chemical shifts are reported in
parts per million (ppm) downfield from tetramethylsilane,
and J values are in Hz. Infrared (IR) spectra were obtained
on an ATI Mattson FT/IR spectrometer. Mass spectra were
recorded using a Waters Micromass ZQ LC-Mass system
and high resolution mass spectra (HRMS) were measured
with a Bruker BioApex FTMS system by direct injection
using an electrospray interface (ESI). When necessary,
chemicals were purified according to the reported proce-
dures (Armarego and Perrin, 1997).
General procedure for the preparation of trans-3,4,5-
trimethoxycinnamamides 10a–e and 11
In the context of our pharmacological research program
dealing with the synthesis of more biologically active
trans-3,4,5-methoxycinnamamides, we wish to report
simple synthesis of trans-3,4,5-methoxycinnamamides and
its derivatives derived from condensation reaction and
coupling reaction. In addition, biologic activities of trans-
3,4,5-methoxycinnamamides and its structural analogs for
antinarcotic activity in mice could suggest us some possi-
ble antinarcotic lead compounds.
To a stirred solution of amide (9, 1.0 g, 4.2 mmol) in dry
dichloromethane (30 mL) 1-ethyl(dimethylaminopropyl)
carbodiimide (EDCI, 0.97 g, 5.0 mmol), 1-hydroxybenzo-
triazole (HOBt, 0.68 g, 5.0 mmol), and triethylamine
(TEA, 0.70 mL, 5.0 mmol) were added and then the
reaction mixture was stirred at room temperature for
30 min. The amines were then added into the reaction
mixture and the resulting mixture stirred at room temper-
ature for 24 h. The reaction mixture was washed with brine
(20 mL) and water (20 mL). The organic layer was sepa-
rated, dried over anhydrous MgSO4, filtered, and the sol-
vent was removed under reduced pressure. The residue was
purified by flash column chromatography (silica gel, ethyl
acetate/hexanes = 2:1, v/v).
Materials and methods
Chemistry
General
(E)-N-2-hydroxyethyl 3,4,5-trimethoxycinnamamide (10a–
e and 11) White solid. mp 135–136 °C. Yield: 60 %;
Rf = 0.6 (dichloromethane/MeOH = 9:1, v/v); IR mmax
(CHCl3, KBr) v 3367 (O–H), 2939 (C–C), 1659 (C=O), 1620
(C=C), 1585, 1551, 1506, 1455, 1420, 1384, 1324, 1281,
Reactions requiring anhydrous conditions were performed
with the usual precautions for rigorous exclusion of air and
moisture. Tetrahydrofuran was distilled from sodium ben-
zophenone ketyl before use. Thin layer chromatography
(TLC) was performed on precoated silica gel G and GP
uniplates from Analtech and visualized with 254 nm UV
1
1243 (C–O), 1218, 1153, 1125, 998, 827, 784 cm-1; H-
NMR (CDCl3, 500.14 MHz) d 7.53 (d, 1H, J = 15.4 Hz),
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Med Chem Res (2013) 22:4615–4621
4617
6.70 (s, 2H), 6.59 (s, 1H), 6.39 (d, 1H, J = 15.4 Hz), 3.86 (s,
3H), 3.84 (s, 6H), 3.73–3.79 (m, 2H), 3.50–3.56 (m, 2H);
13C-NMR (CDCl3, 125.76 MHz) d 167.2, 153.5, 141.4,
139.7, 130.4, 119.9, 105.3, 62.3, 61.0, 56.3, 42.8; LC–MS
(ESI) m/z 304 [M ? Na]?; HRMS: m/z = 282.1334 (calcd.
282.1341 for C14H20NO5: [M?H]?).
(CHCl3, KBr) v 3424, 2923 (C–C), 2837, 1693 (C=O),
1644 (C=C), 1603, 1583, 1505, 1453, 1417, 1341, 1295,
1263, 1212 (C–O), 1187, 1151, 1124, 1047, 1004, 959,
1
824 cm-1; H-NMR (CDCl3, 500.14 MHz) d 7.59 (d, 1H,
J = 15.2 Hz), 6.74 (s, 2H), 6.72 (d, 1H, J = 15.2 Hz),
3.97 (s, 4H), 3.90 (s, 6H), 3.88 (q, 3H, J = 4.5 Hz), 2.69
(dd, 4H, J = 5.2, 4.8 Hz); 13C-NMR (CDCl3,
125.76 MHz) d 165.8, 153.6, 143.5, 140.0, 130.4, 116.3,
105.2, 61.1, 56.4, 45.1, 28.4, 27.5; LC–MS (ESI) m/z 346.34
[M?Na]?; HRMS: m/z = 324.1270 (calcd. 324.1258 for
C16H22NO4S: [M?H]?).
(E)-N-(3-(2-oxopyrrolidin-1-yl)propyl)-3-(3,4,5-trimethox-
yphenyl)acrylamide (10b)
Pale yellow solid. mp
114–115 °C. Yield: 80 %; Rf = 0.6 (MC/MeOH = 10:1,
v/v); IR mmax (CHCl3, KBr) v 3417 (N–H), 3063, 2938 (C–
C), 2837, 1666 (C=O), 1621 (C=C), 1583, 1505, 1452,
1421, 1323, 1279, 1216 (C–O), 1185, 1154, 1124, 1001,
(2E,20E)-1,10-(piperazine-1,4-diyl)bis(3-(3,4,5-trimethoxy-
1
827 cm-1; H-NMR (CDCl3, 500.14 MHz) d 7.52 (d, 1H,
phenyl)prop-2-en-1-one) (11)
White solid. mp 262–
J = 15.6 Hz), 7.14 (t, 1H, J = 6.1 Hz), 6.75 (s, 2H), 6.41
(d, 1H, J = 15.6 Hz), 3.88 (s, 6H), 3.87 (s, 3H), 3.38–3.44
(m, 4H), 3.29–3.37 (m, 2H), 2.45 (t, 2H, J = 7.9 Hz), 2.08
(m, 2H), 1.75 (m, 2H); 13C-NMR (CDCl3, 125.76 MHz) d
176.2, 165.9, 153.4, 140.4, 139.4, 130.6, 120.7, 104.9,
60.9, 56.2, 47.4, 39.5, 35.5, 31.0, 26.4, 18.0; LC–MS (ESI)
m/z 385 [M?Na]?; HRMS: m/z = 363.1931 (calcd.
363.1920 for C19H27N2O5: [M?H]?).
264 °C. Yield: 80 %; Rf = 0.1 (ethyl acetate/n-hex-
ane = 2:1, v/v); IR mmax (CHCl3, KBr) v 3417, 3001, 2939
(C–C), 2838, 1710 (C=O), 1644 (C=C), 1601, 1583, 1505,
1454, 1418, 1337, 1265, 1213 (C–O), 1154, 1124, 1034, 988,
1
822 cm-1; H-NMR (CDCl3, 500.14 MHz) d 7.66 (d, 2H,
J = 15.1 Hz), 6.77 (d, 2H J = 15.1 Hz), 6.76 (s, 4H), 3.92
(s, 12H), 3.87 (s, 6H), 3.84–3.77 (m, 8H); 13C-NMR (CDCl3,
125.76 MHz) d 165.8, 153.6, 144.0, 140.1, 130.7, 115.7,
105.3, 61.1, 56.4; LC–MS (ESI) m/z 549.54 [M?Na]?;
HRMS: m/z = 527.2379 (calcd. 527.2393 for C28H35N2O8:
[M?H]?).
(E)-1-morpholin-4-yl(3,4,5-trimethoxyphenyl)prop-2-en-1-
one (10c) White solid. mp 134–135 °C. Yield: 78 %;
Rf = 0.3 (ethyl acetate/hexanes = 2:1, v/v); IR mmax
(CHCl3, KBr) v 3448, 2852 (C–C), 1645 (C=O), 1587,
1505, 1459, 1341, 1273, 1228 (C–O), 1152, 1127,
1046 cm-1; 1H NMR (CDCl3, 500.14 MHz) d 7.62 (d, 1H,
J = 15.3 Hz), 6.74 (d, 1H, J = 15.3 Hz), 6.75 (s, 2H) 3.90
(s, 6H), 3.88 (s, 3H), 3.73 (s, 8H); 13C NMR (CDCl3,
125.76 MHz) d 165.5, 153.4, 143.3, 139.6, 130.7, 115.7,
105.0, 100.0, 66.9, 61.0, 56.2; LC–MS (ESI) m/z 330.17
[M?Na]?; HRMS: m/z = 308.1485 (calcd. 308.1498 for
C16H22NO5: [M?H]?).
Biologic testing
2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
effects
We used DPPH to test free radical scavenging activity.
DPPH is one of a few stable and commercially available
organic nitrogen radicals and has a UV-via absorption
maximum at 515 nm. Upon reduction, the solution color
fades; the reaction progress is conveniently monitored using
a spectrophotometer. To test the free radical scavenging
effects using DPPH, compounds 10a–e and 11 were
adjusted with methanol solution to final concentration of 10,
50, 1000 lM. Tris–base buffer (0.1 mM) was added and
DPPH radical ethanol solution (1 mL, 0.5 mM) was added
after 5 min. The mixture was warmed in the water bath for
25 min at 37 °C. After 20 min, absorbance was measured
using a spectrophotometer (515 nm). The DPPH radical
scavenging rate of each sample and the 50 % scavenging
concentration based on the DPPH radical scavenging rate
were calculated using the following rate (%).
(E)-1-(4-benzoylpiperazin-1-yl)-3-(3,4,5-trimethoxyphenyl)
prop-2-en-1-one (10d) White solid. mp 179–180 °C.
Yield: 85 %; Rf = 0.7 (MC/MeOH = 10:1, v/v); IR mmax
(CHCl3, KBr) v 3423, 2998 (C–C), 2937 (C–C), 2838,
1721(C=O), 1631 (C=C), 1603, 1582, 1505, 1455, 1421,
1339, 1266, 1219 (C–O), 1153, 1124, 1031, 1007, 923,
1
824 cm-1; H-NMR (CDCl3, 500.14 MHz) d 7.63 (d, 1H,
J = 15.3 Hz), 7.44 (s, 5H), 7.39 (d, 1H, J = 12.8 Hz), 6.75
(s, 2H), 3.90 (s, 6H), 3.88 (s, 3H), 3.74 (s, 8H); 13C-NMR
(CDCl3, 125.76 MHz) d 170.8, 165.8, 153.6, 143.9, 140.0,
135.3, 130.6, 130.2, 128.8, 127.2, 115.8, 105.3, 61.1, 56.4,
53.5; LC–MS (ESI) m/z 433 [M?Na]?; HRMS: m/
z = 411.1912 (calcd. 411.1920 for C23H27N2O5: [M?H]?).
DPPH radical-scavenging rate (%)
A ꢁ C
ꢀ
ꢁ
where A is the absorbance of the sample (DPPH ? com-
¼
1 ꢁ
ꢂ 100
B
(E)-1-thiomorpholino-3-(3,4,5-trimethoxyphenyl)prop-2-en-
1-one (10e) White solid. mp 128–130 °C. Yield: 72 %;
Rf = 0.3 (ethyl acetate/n-hexane = 2:1, v/v); IR mmax
pounds) when a blank was substituted for tris–base buffer,
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Med Chem Res (2013) 22:4615–4621
B is the absorbance of the DPPH radical–ethanol solution
when a blank was substituted for tris–base buffer, and C is
the absorbance of the sample (compounds) alone.
non-neuronal cells. After treatment for 48 h, cells were fed
with fresh media (without fetal calf serum).
Lactate dehydrogenase (LDH) assay
Cortical neuron culture
Lactate dehydrogenases are of great value as in vitro
markers for cellular toxicity. Lactate dehydrogenases
released into the culture medium were measured by mon-
itoring the production of NAD? from NADH during
the conversion of pyruvate to lactate. The cell superna-
tant (30 lL) was incubated in the 120 lL of NADPH
buffer (0.45 mg/mL), after 2 min, followed by addition of
pyruvate (22 mM). The rate of NAD? formation was
monitored for 5 min at 11-s intervals at 340 nm by
spectrophotometer.
Cortical cell cultures were prepared from embryo of the
ICR mice (Daehan Biolink, Eumsung, Korea), gestational
age of 15 days. The cortex was dissected and kept in an
ice-cold solution. The cortical tissues were dissociated to
single cells by a gentle suspension. The cell suspension was
centrifuged at 1,000 rpm for 5 min and then the resulting
pellets were resuspended in the minimal essential media
(MEM), which was supplemented with 5 % heat-inacti-
vated fetal calf serum, mouse serum, glutamine, and glu-
cose. The cells were transferred onto plates coated with
poly-D-lysine and laminin at the density of 4.8 9 105 cells/
well in 24-well cultured plates. The cells were cultured in a
CO2 incubator (5 % [v/v], 37 °C). Seven days after plating,
the cells were treated with 10 lM cytosine arabinofuran-
oside (Ara C) to reduce the growth of contaminating
Animals and drug administration
C57BL/6 mice were obtained from Daehan Biolink
(Eumsung, Korea). They were housed in a 12-h light–dark
cycle and maintained at 24 3 °C. All animal procedures
were in accordance with the Institutional Animal Care and
MeO
MeO
CO2H
MeO
MeO
CHO
MeO
MeO
CO2H
CO2H
b
a
OMe
8
OMe
6
OMe
7
O
O
MeO
MeO
OMe
MeO
MeO
N
R2
d
c
Me
OMe
OMe
9
OH
10a R2=
10b R2=
NH
O
e
NH
N
N
O
OMe
10c R2=
O
MeO
MeO
OMe
OMe
N
O
N
N
N
N
S
10d R2=
10e R2=
OMe
O
11
Scheme 1 Reagents and conditions a CH(CO2H)2, b-alanine, pyri-
dine, reflux, 1.5 h, and conc-HCl, r. t., 1 h (70 %); b 1 N NaOH/
MeOH, r. t., 16 h (73 %); c N,O-dimethylhydroxyamineꢀHCl, Me3Al,
CH2Cl2, 0 °C to r. t., 4 h (85 % from methyl ester) or N,O-
dimethylhydroxyamineꢀHCl, HATU, CH2Cl2, r. t., 2 h (81 % from
acid); d amines, HOBt, EDCI, CH2Cl2, r. t., 2 h (60–85 %);
e piperazine, HATU, CH2Cl2, r. t., 2 h (80 %)
123

Med Chem Res (2013) 22:4615–4621
4619
Use Committee of Ewha Womans University. The mice
(male, 20 2 g) were randomly divided into groups and
given saline, morphine, or both morphine and trans-3,4,5-
trimethoxycinnamamides. The morphine chloride (10 mg/
kg/day, Myungmun Pharm., Seoul) was dissolved in saline
and trans-3,4,5-trimethoxycinnamamides (20 mg/kg/day)
were dissolved in 10 % cremophor solution containing 2 %
dimethyl sulfoxide. Morphine and trans-3,4,5-trimeth-
oxycinnamamides were administered daily for 7 days
intraperitoneally. Trans-3,4,5-trimethoxycinnamamides were
administered 30 min before injection of morphine. Naloxone
hydrochloride (5 mg/kg, i.p.) was injected 6 h after the final
morphine injection for induction of morphine withdrawal
syndrome in mice.
diyl)-bis-[3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one] (11)
in 80 % yield.
Biology
Radical scavenging activity
DPPH radicals are a useful method for the preliminary
screening of compounds capable of scavenging activated
oxygen species since these nitrogen radicals are much more
stable and easier to handle than oxygen free radicals. The
radical scavenging activities of the trans-3,4,5-trimeth-
oxycinnamamides were evaluated by an established test
method (Lawinski et al., 2005) over the concentration
range of 10, 50, and 1000 lM (Fig. 2). Scavenging effects
were found for almost all the compounds 10a–e over 20 %
at the higher concentration although 1 mM actions were
less effective than the reference material, resveratrol. We
have found that the piperazine based trans-3,4,5-trimeth-
oxycinnamamides possessed relatively higher antioxidant
activity than morpholin-based trans-3,4,5-trimethoxycinn-
amamides in vitro.
Measurement of antinarcotic behavior on morphine
Morphine withdrawal syndrome was induced by the
injection of naloxone (5 mg/kg), which is a competitive
antagonist with high opioid receptor affinity. Immediately
after the naloxone injection, mice were placed into the
individual observation cylinders (24 cm in diameter and
50 cm in high) and the frequency of jumps of each mouse
was observed for 30 min.
Cell viability assay
Lactate dehydrogenases are valuable in vitro markers for
cellular toxicity. In these studies, LDH activity was used as
a measure of the neuroprotective effects of trans-3,4,5-
trimethoxycinnamamide compounds. The trans-3,4,5-tri-
methoxycinnamamide derivatives 10a, 10d, and 10e
showed the most potent neuroprotective activity in gluta-
mate-induced primary cortical neuronal cells at the rela-
tively low doses (5–20 lM; Fig. 3).
Results and discussion
Chemistry
The preparation of trans-3,4,5-trimethoxycinnamamides
10a–e and 11 was initiated from commercially available
3,4,5-trimethoxyaldehyde (6) as a starting material, treated
with malonic acid and b-alanine in pyridine to give diacid
7, and readily decarboxylated in the presence of acidic
media to generate acid 8 in 70 % yield in two steps (Stabile
and Dicks, 2004). Compound 7 was also treated with basic
media as 1 N sodium hydroxide in methanol to yield acid 8
in 73 % yield. Compound 8 was treated with N,O-dime-
thylhydroxyamine-mono hydrochloride salt in the presence
of Me3Al in dichloromethane to give amide 9 in 85 %
yield. Acid 8 also was directly transferred to give amide 9
under coupling condition [(2-(7-aza-1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),
diisopropylamine (DIPEA) in dichloromethane) in 81 %
yield. Amide 9 was treated with various amines through
coupling condition [ethyl(dimethylaminopropyl)carbodi-
imide (EDCI), 1-hydroxybenzotriazole (HOBt), and
triethylamine (TEA) in dichloromethane to generate trans-
3,4,5-trimethoxycinnamamides 10a–e in 60–85 % yields
(Scheme 1).
80
10uM
50uM
1mM
60
40
20
0
10a
10b
10c
10d
10e
11
resver
Fig. 2 Inhibitory actions of trans-3,4,5-trimethoxycinnamamides
10a–e and 11 on scavenging of DPPH radical with different
concentrations (10, 50, and 1000 lM). Data represent the
mean standard error of three observations
Interestingly, the coupling of amide 9 with HATU/
CH2Cl2 to produce dimmer (2E, 20E)-1,10-(piperazine-1,4-
123

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Med Chem Res (2013) 22:4615–4621
120
5uM
trans-3,4,5-trimethoxycinnamamides. All of trans-3,4,5-
trimethoxycinnamamides significantly inhibited the jump-
ing frequencies in morphine-dependent mice. We found that
trans-3,4,5-trimethoxycinnamamides attenuated jumping
frequency effectively about 80–90 % after naloxone injec-
tion. Interestingly, compound 11 which contains two tri-
methoxyphenyl groups showed a very strong antinarcotic
effect. There is an interesting report that sinapic acid which
contains two methoxyl groups and one hydroxyl group
instead of three hydroxyl groups on the aromatic ring
showed anxiolytic-like effect in mice (Yoon et al., 2007).
This result suggests that sinapic acid may have an antinar-
cotic effect.
10uM
20uM
100
80
60
40
20
0
*
*
*
*
*
*
10a
10b
10c
10d
10e
11 glut 50uM
Fig. 3 Protective actions of trans-3,4,5-trimethoxycinnamamides
10a–e and 11 on glutamate-induced neurotoxicity in cultured cortical
neurons with different concentrations (5, 10, and 20 lM). Compounds
were co-treated with excitatory amino acid, glutamate (50 lM) for
24 h at 37 °C. Data represent the mean standard error of three
observations. *p \ 0.05 in comparison with glutamate only group
Conclusions
We have prepared trans-3,4,5-trimethoxycinnamamides
10a–e and 11 from inexpensive and readily available
materials and evaluated their biologic activities. We have
found that the piperazine-based trans-3,4,5-trimethoxyc-
innamamides showed inhibitory activity on neurotoxicity
in vitro. Interestingly, trans-3,4,5-trimethoxycinnamamides
10a–e and 11 showed very strong inhibitory activity on the
morphine withdrawal syndrome in mice. We conclude that
simple synthesis of trans-3,4,5-trimethoxycinnamamides
and key fragments are useful for the development of anti-
narcotic agents for the treatment of morphine withdrawal
syndrome.
Antinarcotic properties
The mice received morphine (10 mg/kg/day, i.p.) for 7 days
to develop dependence on morphine and examined the
effects of trans-3,4,5-trimethoxycinnamamides (20 mg/kg/
day, i.p.) on naloxone-induced jumping behavior in mor-
phine-dependent mice. As shown in Fig. 4, jumping fre-
quencies as indicator of morphine withdrawal symptom
were strongly decreased by treatment with TMCA 8 and
Acknowledgments This research was supported by the National
Research Foundation of Korea (NRF) Grant funded by the Korea
Government (MEST) (MRC, 2010-0029355).
100
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