Synthesis and Evaluation of a Series of 3,4,5-Trimethoxycinnamic Acid Derivatives as Potential Antinarcotic Agents

Article abstract of DOI:10.1111/cbdd.12087

A series of 3,4,5-trimethoxycinnamic acid derivatives was prepared and evaluated for antinarcotic effects on morphine dependence in mice and binding affinities on serotonergic receptors. The key synthetic strategies involve generation of ketones 6-7, esters 9-12 through condensation reaction, and amides 13-19 via coupling reaction using 1-hydroxybenzotriazole/ethyl(dimethylaminopropryl)carbodiimide system in high yield. We found that the naloxone-induced morphine withdrawal syndrome was significantly suppressed by new synthetic 3,4,5-trimethoxycinnamic acid derivatives (20mg/kg/day). Most of 3,4,5-trimethoxycinnamic acid derivatives were found to have high affinity to 5-HT_1A receptor. The naloxone-induced morphine withdrawal syndrome was attenuated by (+)8-OH-DPAT (0.1mg/kg/day, i.p.), a 5-HT_1A receptor agonist. In cortical neuronal cells, (+)8-OH-DPAT (1μm) produced an elevation of the pERK 1/2 expression, and the elevated pERK levels were inhibited by WAY 100635, a 5-HT_1A receptor-specific antagonist. Interestingly, the pERK levels were increased by the 3,4,5-trimethoxycinnamic acid derivatives and the derivatives-mediated changes in pERK levels were blocked by the WAY 100635. These results suggested that new synthetic 3,4,5-trimethoxycinnamic acid derivatives have a potential antinarcotic effect through acting as a 5-HT_1A receptor agonist in mice.

Full text of DOI:10.1111/cbdd.12087

Chem Biol Drug Des 2013; 81: 389–398
Research Article
Synthesis and Evaluation of a Series of
3,4,5-Trimethoxycinnamic Acid Derivatives as Potential
Antinarcotic Agents
Jae-Chul Jung1, Sohyeon Moon1, Dongguk Min2,
Methoxycinnamic acids or hydroxycinnamic acids are an
important substance for the remediation of serious disease,
many of which possess widespread pharmacological prop-
erties related to various therapeutic fields such as oncology
Woo Kyu Park3, Mankil Jung2,* and Seikwan
Oh1,*
1Department of Neuroscience and TIDRC, School of
(1), osteoporosis (2), arthritis (3), anti-inflammatory (4), and
inhibition of enzymes (5). Current research priorities are to
better characterize the biological roles and biochemical fea-
tures of trans-cinnamic acid derivatives (Figure 1).
Medicine, Ewha Womans University, Yangchon-Ku, Seoul
158-710, Korea
2Department of Chemistry, Yonsei University, Seodaemun-
Ku, Seoul 120-749, Korea
3Center for Drug Discovery Technologies, Korea Research
Institute of Chemical Technology, Yuseong-Gu, Daejon
305-343, Korea
In particular, 3,4,5-trimethoxycinnamic acid (TMCA) and its
derivatives were recently focused on the role mechanism
of anti-oxidative processes (6) and directed to the anti-
oxidant activity as well as neurological disorders (7). Accu-
mulated publications have focused on the stress induced
with repeated cold exposure or intracerebroventricular
injection of corticotrophin-releasing hormone (8). Recently,
the Kawashima group (9) reported TMCAs were having
significant anti-stress effects by suppressing norepineph-
rine (NE) content in locus coeruleus (LC). Interestingly, it
has been known that elevation of NE in LC was shown in
morphine withdrawal syndrome. The Geraci group (10)
developed the hydroxycinnamic acid clustered by a calixa-
rene platform for the radical scavenging and anti-oxidant
activity. The Keung group (11) described the eugenol and
its structural analogues exhibit antidepressant-like activity.
*Corresponding authors: Mankil Jung, mjung@yonsei.ac.kr;
Seikwan Oh, skoh@ewha.ac.kr
A series of 3,4,5-trimethoxycinnamic acid derivatives
was prepared and evaluated for antinarcotic effects on
morphine dependence in mice and binding affinities on
serotonergic receptors. The key synthetic strategies
involve generation of ketones 6–7, esters 9–12 through
condensation reaction, and amides 13–19 via coupling
reaction using 1-hydroxybenzotriazole ⁄ ethyl(dimethyla-
minopropryl)carbodiimide system in high yield. We
found that the naloxone-induced morphine withdrawal
syndrome was significantly suppressed by new syn-
thetic
3,4,5-trimethoxycinnamic
acid
derivatives
(20 mg ⁄ kg ⁄ day). Most of 3,4,5-trimethoxycinnamic acid
derivatives were found to have high affinity to 5-HT1A
receptor. The naloxone-induced morphine withdrawal
syndrome was attenuated by (+)8-OH-DPAT (0.1 mg ⁄
kg ⁄ day, i.p.), a 5-HT1A receptor agonist. In cortical neu-
ronal cells, (+)8-OH-DPAT (1 lM) produced an elevation
of the pERK 1 ⁄ 2 expression, and the elevated pERK lev-
els were inhibited by WAY 100635, a 5-HT1A receptor-
specific antagonist. Interestingly, the pERK levels were
increased by the 3,4,5-trimethoxycinnamic acid deriva-
tives and the derivatives-mediated changes in pERK
levels were blocked by the WAY 100635. These results
suggested that new synthetic 3,4,5-trimethoxycinnamic
acid derivatives have a potential antinarcotic effect
through acting as a 5-HT1A receptor agonist in mice.
Morphine is most frequently used as analgesics in clinical
treatment for reducing the sustaining pain. Chronic opioids
exposure induces a strong dependence state and induce
withdrawal syndrome when it was stopped abruptly. Injec-
tion of a general opioid receptor antagonist naloxone after
a continuous opioid treatment leads to a withdrawal syn-
drome as characteristic behavior of opioid dependence
(12). It has been reported that opioids have their depen-
dence as well as analgesic effect through several pathways
such as the serotonergic (13), glutamatergic (14), dopami-
nergic (15), GABAergic (16), and noradrenergic pathways
(17). The accumulation of 5-hydroxytryptophan (5-HTP)
and 3,4-dihydroxyphenylalanine (DOPA), a precursor of
serotonin and dopamine, was increased by chronic mor-
phine treatment and during morphine withdrawal syndrome
precipitated by naloxone in various brain regions (18).
Key words: binding affinity, coupling reaction, morphine,
pERK, 3,4,5-trimethoxycinnamic acids, 5-HT1A receptor
Received 22 July 2012, revised 28 September 2012 and
accepted for publication 24 October 2012
Hyperactivity of LC neuron is an important state of opiate
withdrawal. LC hyperactivity by opiate withdrawal is medi-
ª 2012 John Wiley & Sons A/S. doi: 10.1111/cbdd.12087
389

Jung et al.
Figure 1: Structures of various cinnamic acids 1–4.
ated by an excitatory amino acid release to the LC. 5-HT
selectively blocks the way of excitatory amino acid release
to the LC during opiate withdrawal (19). In addition, it has
been reported the role of 5-HT (5-hydroxytryptamine) in
the development of dependence on opiates (20), these
studies indicated that 5-HT system may be related with
opiate pathway in central nervous system (CNS) and
hyperactivity of LC neuron induced by opiate withdrawal
was deceased by activation of serotonergic neurotransmis-
sion. Several selective 5-HT1A agonists include (+)8-OH-
DPAT [8-hydroxy-2-(di-n-propylamino)tertraline] attenuate
the antinociception of l-opioid agonist, morphine in mice,
but j-opioid agonist action was not antagonized by 5-
HT1A agonist (21,22). 5-HT1A receptor agonist induced
activation of extracellular signal-regulated kinase (ERK1 ⁄ 2)
and Akt for survival. (+)8-OH-DPAT stimulated ERK1 ⁄ 2 in
hypothalamus of rat in time-dependent manner (23).
date ⁄ sulfuric acid solution followed by heating on a hot
plate. Flash column chromatography was performed with
Merck silica gel 60 (230–400 mesh) or purification of reac-
tion mixture was recrystallized by diethyl ether in dichlo-
1
romethane. H NMR and 13C NMR spectra were recorded
on Bruker DRX 250 at 250 and 63 MHz or 400 at 400 and
100 MHz, respectively. Proton chemical shifts are reported
in ppm (d) relative to internal tetramethylsilane (TMS, d
0.00) or with the solvent reference relative to TMS
employed as the internal standard (CDCl3, d 7.26 ppm).
Data are reported as follows: chemical shift {multiplicity
[singlet (s), doublet (d), triplet (t), quartet (q), and multiplet
(m)], coupling constants [Hz], integration}. Carbon chemical
shifts are reported in ppm (d) relative to TMS with the
respective solvent resonance as the internal standard
(CDCl3, d 77.16 ppm). Infrared (IR) spectra were recorded
on a JASCO FT ⁄ IR-430 spectrometer. Data are reported in
wave numbers (per cm). ESI-LC-MS was recorded on a
Waters ZQ 4000 LC-MS spectrometer. Melting Points were
determined on a BIBBY Stuart Scientific MELTING POINT
APPRATUS SMP3. Elemental analyses were performed on
a CE instrument EA1110 (25).
In the context of our medicinal chemistry program dealing
with the development of TMCA and its derivatives having
remarkable bioactivities for the neurological disorders, we
have prepared fourteen TMCA derivatives. Previously, we
have reported that 3,4,5-trimethoxyphenyl acrylamides
showed good antinarcotic effect in mice (24). We wish to
report herein a simple synthesis and evaluation of biologi-
cal activity of TMCA derivatives 6–19 during morphine
withdrawal in vivo, starting from TMCA via chlorination,
esterification, and global coupling reaction. It was deter-
mined whether TMCA derivatives can attenuate the nalox-
one-induced jumping behavior in morphine-dependent
mice, and to identify what is the main target among sev-
eral neurotransmitter pathways and receptors being medi-
ated in CNS.
(E)-N-methoxy-N-methyl 3,4,5-trimethoxycinnama
mide (6)
To a stirred solution of TMCA (3, 1.0 g, 4.2 mmol) in dry
dichloromethane (30 mL) was added ethyl(dimethylamino-
propryl)carbodiimide (EDCI, 0.97 g, 5.0 mmol), 1-hydrox-
ybenzotriazole (HOBt, 0.68 g, 5.0 mmol), and triethylamine
(TEA, 0.70 mL, 5.0 mmol) and then the mixture was stirred
at room temperature for 30 min. The N,O-dimethylhydr-
oxyamine (0.49 g, 5.0 mmol) was added to the reaction at
room temperature and the resulting mixture was stirred at
room temperature for 24 h. The reaction mixture was
diluted with dichloromethane (10 mL) and washed with
saturated aqueous NH4Cl solution (30 mL) and brine
(30 mL). The organic layer was separated, dried over
anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash col-
umn chromatography (silica gel, ethyl acetate ⁄ n-hex-
anes = 2:1, v ⁄ v) to give pure 6 as white solid (0.89 g,
75%). White solid. mp 106–107 ꢀC (lit (26). mp 102–
104 ꢀC); Rf = 0.4 (ethyl acetate ⁄ n-hexanes = 2:1, v ⁄ v); IR
mmax (CHCl3, KBr) 3466, 2945, 1650, 1613, 1583, 1505,
1459, 1420, 1385, 1333, 1246, 1153, 1127, 1005 per
cm; 1H NMR (250 MHz, CDCl3) d 7.66 (d, 1H, J = 15.7),
Experimental Section
Chemistry
All other commercial reagents and solvents were used as
received without further purification. Reaction solvents were
distilled from calcium hydride for dichloromethane and from
sodium metal and benzophenone for tetrahydrofuran. The
reactions were monitored and the Rf values determined
using analytical thin layer chromatography (TLC) with Merck
silica gel 60, F-254 precoated plates (0.25 mm thickness).
Spots on the TLC plates were visualized using ultraviolet
light (254 nm),
a cerium sulfate ⁄ ammonium dimolyb-
390
Chem Biol Drug Des 2013; 81: 389–398

3,4,5-Trimethoxycinnamic Acid Derivatives
6.92 (s, 2H), 6.79 (d, 1H, J = 15.7), 3.91 (s, 6H), 3.88 (s,
3H), 3.78 (s, 3H), 3.32 (s, 3H); 13C NMR (63 MHz, CDCl3)
d 166.9 153.4 143.7 139.8 130.8 115.0 105.3 62.0 61.0
56.3 32.6; LC-MS (ESI+) m ⁄ z 321.47 [M+Na].
1311, 1279, 1245, 1127, 1003 per cm; 1H NMR
(250 MHz, CDCl3) d 7.61 (d, 1H, J = 15.9), 6.75 (s, 2H),
6.36 (d, 1H, J = 15.9), 3.88 (s, 9H), 3.80 (s, 3H); 13C NMR
(63 MHz, CDCl3) d 167.3 153.3 144.8 140.0 129.8 116.9
105.1 60.9 56.0 51.6; LC-MS (ESI+) m ⁄ z 275.02 [M+Na].
Preparation of compounds 7 and 8
To a solution of 6 (0.5 g, 1.8 mmol) in dry THF (20 mL)
was added dropwise Et-MgBr (0.48 g, 3.6 mmol) or Ph-
MgBr (0.65 g, 3.6 mmol) at )78 ꢀC and the mixture was
warmed to room temperature for 2.5 h. The reaction mix-
ture was quenched by slowly addition of 10% aqueous
HCl solution (two drops) and washed with brine (15 mL).
The organic layer was separated, dried over anhydrous
MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by flash column chromatog-
raphy (silica gel, ethyl acetate ⁄ n-hexanes = 2:1, v ⁄ v) to
give pure 7 and 8.
(E)-Ethyl 3,4,5-trimethoxycinnamate (10)
White solid. mp 69–70 ꢀC (lit (29). mp 68–69 ꢀC); Rf = 0.7
(ethyl acetate ⁄ n-hexanes = 2:1, v ⁄ v); IR mmax (CHCl3, KBr)
3464, 2353, 1710, 1635, 1582, 1506, 1456, 1416, 1275,
1152, 1126, 1003 per cm; 1H NMR (250 MHz, CDCl3) d
7.61 (d, 1H, J = 15.9), 6.76 (s, 2H), 6.35 (d, 1H, J = 15.9),
4.27 (q, 2H), 3.89 (s, 6H), 3.88 (s, 3H), 1.35 (t, 3H); 13C
NMR (63 MHz, CDCl3) d 167.1 153.6 144.7 142.0 130.1
117.6 105.3 61.1 60.7 56.3 14.5; LC-MS (ESI+) m ⁄ z
289.07 [M+Na].
(E)-Phenyl 3,4,5-trimethoxycinnamate (11)
(E)-Ethyl 3,4,5-trimethoxycinnamyl ketone (7)
Pale beige solid. mp 110–112 ꢀC; Rf = 0.7 (ethyl ace-
tate ⁄ n-hexanes = 2:1, v ⁄ v); IR mmax (CHCl3, KBr) 2933,
1661, 1625, 1614, 1581, 1504, 1461, 1420, 1336, 1245,
1194, 1156, 1127 per cm; 1H NMR (250 MHz, CDCl3) d
7.49 (d, 1H, J = 16.1), 6.78 (s, 2H), 6.66 (d, 1H, J = 16.1),
3.91 (s, 6H), 3.89 (s, 3H), 2.72 (q, 2H), 1.18 (t, 3H); 13C
NMR (63 MHz, CDCl3) d 201.0 153.6 142.4 140.4 130.2
125.5 105.6 61.1 56.3 34.0 8.4; LC-MS (ESI+) m ⁄ z
273.22 [M+Na]. Anal. Calcd for (C14H18O4): C, 67.18; H,
7.25. Found: C, 67.18; H, 7.26.
To a solution of TMCA (4, 1.0 g, 4.2 mmol) was added
thionyl chloride (0.37 mL, 5.0 mmol) and the mixture was
heated at 65 ꢀC on a water bath, until hydrogen chloride
ceases to be evolved (about 1 h). The generated acid
chloride 5 was used without purification. The phenol
(0.44 mL 5.0 mmol) was added to reaction mixture at
65 ꢀC and the resulting mixture was heated at 65 ꢀC on a
water bath until the hydrogen chloride was not evolved for
approximately 1 h. The reaction mixture was allowed to
cool at room temperature and distilled under reduced
pressure. The residue was by flash column chromatogra-
phy (silica gel, ethyl acetate ⁄ n-hexanes = 1:6, v ⁄ v) to give
pure 11. White solid. mp 103–104 ꢀC; Rf = 0.2 (ethyl ace-
tate ⁄ n-hexanes = 1:6, v ⁄ v); IR mmax (CHCl3, KBr) 3468,
2842, 1726, 1635, 1582, 1503, 1455, 1420, 1272, 1244,
1194, 1131, 1005 per cm; 1H NMR (250 MHz, CDCl3) d
7.79 (d, 1H, J = 15.9), 7.15–7.44 (m, 5H), 6.82 (s, 2H),
6.55 (d, 1H, J = 15.9), 3.90 (s, 9H); 13C NMR (63 MHz,
CDCl3) d 165.5 153.6 150.9 146.6 140.5 129.7 129.5
125.9 121.7 116.6 105.5 61.1 56.3; LC-MS (ESI+) m ⁄ z
337.02 [M+Na].
(E)-Phenyl 3,4,5-trimethoxycinnamyl ketone (8)
Pale yellow solid. mp 138–139 ꢀC (lit (27). mp 135–
136 ꢀC); Rf = 0.8 (ethyl acetate ⁄ n-hexanes = 2:1, v ⁄ v); IR
mmax (CHCl3, KBr) 2923, 1603, 1450, 1413, 1385, 1124,
1096, 1080, 1041 per cm; 1H NMR (400 MHz, CDCl3) d
8.00–8.02 (m, 2H) 7.72 (d, 1H, J = 15.6), 7.58–7.62 (m,
1H), 7.50–7.54 (m, 2H), 7.40 (d, 1H, J = 15.6), 6.87 (s,
2H), 3.93 (s, 6H), 3.91 (s, 3H); 13C NMR (63 MHz, CDCl3)
d 190.7 153.5 145.2 138.3 132.9 130.4 128.8 128.6
121.5 105.5 61.2 56.3; LC-MS (ESI+) m ⁄ z 321.28 [M+Na].
(E)-Benzyl 3,4,5-trimethoxycinnamate (12)
To a solution of TMCA (3, 1.0 g, 4.2 mmol) and benzyl
bromide (0.60 mL, 5.0 mmol) in 30ml of DMF ⁄ 1,4-Dioxane
(1:1), NaHCO3 (0.42 g, 5.0 mmol) was added at room
temperature. The reaction mixture was heated and stirred
at 90 ꢀC for 24 h. The mixture was cooled to room tem-
perature and diluted with ethyl acetate. And then it was
washed with brine and water. The organic layer was dried
over anhydrous MgSO4, filtered, and the solvent was
removed under reduced pressure. The residue was puri-
fied by flash column chromatography (silica gel, ethyl ace-
tate ⁄ n-hexanes = 2:1, v ⁄ v) to give pure 12. White solid.
mp 92–93 ꢀC; Rf = 0.7 (ethyl acetate ⁄ n-hexanes = 2:1,
v ⁄ v); IR mmax (CHCl3, KBr) 3436, 2945, 1713, 1638, 1583,
1506, 1455, 1417, 1276, 1244, 1149, 1126, 1002 per
Preparation of compounds 9 and 10
The solution of TMCA (9, 1.0 g, 4.2 mmol) and H2SO4 (2–
3 drops) in methanol (15 mL) or ethanol (15 mL) was ref-
luxed for 24 h. The mixture was cooled to room tempera-
ture and the solid was filtered. The crystal was washed
with ether (5 mL) and dried under reduced pressure to
generate 9 and 10.
(E)-Methyl 3,4,5-trimethoxycinnamate (9)
White solid. mp 101–102 ꢀC (lit (28). mp 91.5–92 ꢀC);
Rf = 0.9 (ethyl acetate ⁄ n-hexanes = 2:1, v ⁄ v); IR mmax
(CHCl3, KBr) 3444, 3026, 1708, 1636, 1584, 1504, 1416,
Chem Biol Drug Des 2013; 81: 389–398
391

Jung et al.
cm; 1H NMR (250 MHz, CDCl3) d 7.65 (d, 1H, J = 15.9),
7.32–7.47 (m, 5H), 6.75 (s, 2H), 6.40 (d, 1H, J = 15.9),
5.25 (s, 2H), 3.88 (s, 9H); 13C NMR (63 MHz, CDCl3) d
166.9 153.5 145.2 140.2 136.1 129.9 128.7 128.4 117.2
105.3 66.5 61.1 56.2; LC-MS (ESI+) m ⁄ z 351.20 [M+Na].
J = 15.5), 6.25–6.32 (m, 3H), 4.52 (d, 2H, J = 13.9), 3.85
(s, 6H), 3.83 (s, 3H); 13C NMR (63 MHz, CDCl3) d 165.7
153.4 151.3 142.3 141.4 139.6 130.4 119.8 110.6 107.7
105.0 61.0 56.1 36.7; LC-MS (ESI+) m ⁄ z 340.01 [M+Na].
(E)-1-Morpholin-4-yl(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (16)
General procedure for the preparation of amides
13–19
White solid. mp 134–135 ꢀC (lit (31). mp 129–131 ꢀC);
Rf = 0.3 (ethyl acetate ⁄ hexanes = 2:1, v ⁄ v); IR mmax
(CHCl3, KBr) 3448, 2852, 1645, 1587, 1505, 1459, 1341,
1273, 1228, 1152, 1127, 1046 per cm; 1H NMR
(250 MHz, CDCl3) d 7.62 (d, 1H, J = 15.3), 6.74 (d, 1H,
J = 15.3), 6.75 (s, 2H) 3.90 (s, 6H), 3.88 (s, 3H), 3.73 (s,
8H); 13C NMR (63 MHz, CDCl3) 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].
To a stirred solution of TMCA (9, 1.0 g, 4.2 mmol) in dry
dichloromethane (30 mL) was added EDCI (0.97 g,
5.0 mmol), HOBt (0.68 g, 5.0 mmol), and triethylamine
(TEA, 0.70 mL, 5.0 mmol) and then the reaction mixture
was stirred at room temperature for 30 min. Each amine
was then added into the reaction mixture and the resulting
mixture was stirred at room temperature for 24 h. The
reaction mixture was washed with brine (20 mL) and water
(20 mL). The organic layer was separated, dried over
anhydrous MgSO4, filtered, and the solvent was removed
under reduced pressure. The residue was purified by flash
column chromatography (silica gel, ethyl acetate ⁄ hex-
anes = 2:1, v ⁄ v).
(E)-1-[4-(3,5-Dimethoxyphenyl)piperazin-1-yl]-3-(3,4,
5-trimethoxyphenyl)prop-2-en-1-one (17)
Yellow solid. mp 168–170 ꢀC; Rf = 0.5 (ethyl acetate ⁄ hex-
anes = 2:1, v ⁄ v); IR mmax (CHCl3, KBr) 3466, 2943, 1646,
1504, 1463, 1199, 1150, 1127, 1041 per cm; 1H NMR
(250 MHz, CDCl3) d 7.62 (d, 1H, J = 15.3) 6.82 (d, 1H,
J = 15.4) 6.76 (s, 2H) 6.10 (d, 2H, J = 1.9) 6.06 (d, 1H,
J = 1.8) 3.90 (s, 6H) 3.88 (s, 3H) 3.83 (s, 4H) 3.78 (s, 6H)
3.22 (s, 4H); 13C NMR (63 MHz, CDCl3) d 165.4 161.5
153.4 152.8 143.2 139.6 130.8 116.1 105.0 95.5 92.1
61.0 56.2 55.3 49.6; LC-MS (ESI+) m ⁄ z 465.17 [M+Na].
Anal. Calcd for (C24H30N2O6): C, 65.14; H, 6.83; N, 6.33.
Found: C, 65.14; H, 6.83; N, 6.35.
(E)-1-(Pyrrolidin-1-yl)-3-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (13)
White solid. mp 159–160 ꢀC (lit (30). mp 148–150 ꢀC);
Rf = 0.2 (ethyl acetate ⁄ hexanes = 2:1, v ⁄ v); IR mmax
(CHCl3, KBr) 3469, 2970, 1646, 1584, 1507, 1415, 1332,
1243, 1128, 1004 per cm; 1H NMR (250 MHz, CDCl3) d
7.62 (d, 1H, J = 15.4), 6.75 (s, 2H), 6.62 (d, 1H, J = 15.4),
3.90 (s, 6H), 3.88 (s, 3H), 3.58–3.69 (m, 4H), 1.68–2.05
(m, 4H); 13C NMR (100 MHz, CDCl3) d 164.8 153.5 142.0
131.1 118.2 105.2 61.0 56.4 46.8 46.2 26.3 24.5; LC-MS
(ESI+) m ⁄ z 314.09 [M+Na].
(E)-1-(4-Benzylpiperidin-1-yl)-3-(3,4,5-trimethoxy
phenyl)prop-2-en-1-one (18)
White solid. mp 120–122 ꢀC; Rf = 0.5 (ethyl acetate ⁄ hex-
anes = 2:1, v ⁄ v); IR mmax (CHCl3, KBr) 3466, 2209, 1645,
1456, 1125 per cm; H NMR (250 MHz, CDCl3) d 7.55 (d,
(E)-N-(Tetrahydrofuran-2-yl)methyl 3,4,5-trimethoxyc
innamamide (14)
1
White solid. mp 98–99 ꢀC; Rf = 0.2 (ethyl acetate ⁄ hex-
anes = 2:1, v ⁄ v); IR mmax (CHCl3, KBr) 3428, 1631, 1545,
1505, 1419, 1321, 1281, 1125 per cm; 1H NMR
(250 MHz, CDCl3) d 7.51 (d, 1H, J = 15.5), 6.85–6.67 (m,
3H), 6.45 (d, 1H, J = 15.5), 4.27–3.95 (m, 2H), 3.85 (s,
9H), 3.80–3.59 (m, 2H), 3.37–3.20 (m, 1H), 2.11–1.81 (m,
3H), 1.70–1.55 (m, 1H); 13C NMR (63 MHz, CDCl3) d
166.2 153.2 140.8 139.2 130.4 120.0 104.7 77.68 67.9
60.8 55.9 43.4 28.6 25.7; LC-MS (ESI+) m ⁄ z 344.15
[M+Na].
1H, J = 15.3), 7.32–7.21 (m, 5H), 6.78 (d, 1H, J = 15.3),
6.73 (s, 2H), 4.74–4.61 (m, 1H), 4.17–4.05 (m, 1H), 3.89
(s, 6H), 3.87 (s, 3H), 3.05 (t, 1H, J = 12.2), 2.63–2.51 (m,
2H), 1.74 (d, 3H, J = 12.4), 1.30–1.14 (m, 2H); 13C NMR
(63 MHz, CDCl3) d 165.3 153.4 142.4 139.9 139.4 131.1
129.1 128.3 126.1 116.9 104.9 61.0 56.2 46.3 43.0 38.4
31.9; LC-MS (ESI+) m ⁄ z 418.10 [M+Na].
(E)-N-4-ethylphenyl 3,4,5-trimethoxycinnamamide
(19)
Pale yellow solid. mp 135–136 ꢀC; Rf = 0.1 (ethyl ace-
tate ⁄ hexanes = 2:1, v ⁄ v); IR mmax (CHCl3, KBr) 3247,
1661, 1585, 1504, 1462, 1415, 1330, 1276, 1129, 1007
per cm; 1H NMR (250 MHz, CDCl3) d 7.65 (d, 2H,
J = 15.4), 7.54 (d, 2H, J = 8.0), 7.17 (d, 2H, J = 8.3), 6.73
(s, 2H), 6.51 (d, 1H, J = 15.4), 3.88 (s, 3H), 3.85 (s, 6H),
2.62 (q, 2H), 1.22 (t, 3H); 13C NMR (63 MHz, CDCl3) d
165.7 153.1 141.4 140.3 139.2 136.1 130.3 128.2 120.9
(E)-N-(Furan-2-ylmethyl) 3,4,5-trimethoxycinnamamide
(15)
Pale yellow solid. mp 133–134 ꢀC; Rf = 0.6 (ethyl ace-
tate ⁄ hexanes = 2:1, v ⁄ v); IR mmax (CHCl3, KBr) 3449,
2945, 1661, 1620, 1583, 1503, 1458, 1421, 1322, 1281,
1
1151, 1124 per cm; H NMR (250 MHz, CDCl3) d 7.56 (d,
1H, J = 15.5), 7.34 (s, 1H), 6.70 (s, 2H), 6.38 (d, 1H,
392
Chem Biol Drug Des 2013; 81: 389–398

3,4,5-Trimethoxycinnamic Acid Derivatives
120.1 104.8 60.8 55.7 28.2 15.5; LC-MS (ESI+) m ⁄ z
364.14 [M+Na].
For serotonin 5-HT2A receptor binding, an aliquot of frozen
membrane from CHO-K1 cell line expressing the human
recombinant 5-HT2A receptor (PerkinElmer) and [3H]Ke-
tanserin (1 nM; PerkinElmer) were used in the presence of
mianserin (20 lM) as non-specific. The reaction mixture
was incubated for 60 min at 27 ꢀC using 50 mM Tris–HCl
(pH 7.4) buffer containin 4 mM CaCl2 and 0.1% ascorbic
acid, and harvested through filtermat A glass fiber filter
presoaked in 0.5% PEI.
Biological testing
Animals and drug administration
C57BL ⁄ 6 mice were obtained from Daehan Biolink (Eum-
sung, Korea). It was housed on a 12 h light-dark cycle
and maintained at 24 3 ꢀC. All animal procedures were
in accordance with the Institutional Animal Care and Use
Committee of Ewha Womans University. The mice (male,
20 2 g) were randomly divided into each group and
were given saline, morphine, or both morphine and TMCA
analogues. The morphine chloride (10 mg ⁄ kg ⁄ day, Myung-
mun Pharm., Seoul) was dissolved in saline and TMCA
analogues (20 mg ⁄ kg ⁄ day) were dissolved in 10% cremo-
phor solution containing 2% dimethyl sulfoxide. Morphine
and TMCA analogues were administered daily for 7 days
intraperitoneally. TMCA analogues were administered
30 min prior to injection of morphine. Naloxone hydrochlo-
ride (5 mg ⁄ kg, i.p.) was injected 6 h after the final mor-
phine injection for induction of morphine withdrawal
syndrome in mice.
For serotonin 5-HT2C receptor binding assays, frozen
membranes from stable CHO-K1 cell line expressing the
human recombinant 5-HT2C receptor (PerkinElmer), 1 nM
[3H]Mesulergine (Amersham Biosciences, Buckingham-
shire, UK) and test compounds were added into 50 mM
Tris–HCl (pH 7.4) buffer containing 0.1% ascorbic acid
and 4 mM CaCl2. Non-specific binding was determined
using 100 lM mianserin. The incubations were performed
for 60 min at 27 ꢀC, and these were terminated by rapid
filtration through filtermat A glass fiber filter presoaked in
0.5% PEI.
For serotonin 5-HT6 receptor binding, frozen membrane
from stable CHO-K1 cell line expressing the human
recombinant 5-HT6 receptor (PerkinElmer), 1 nM [3H]LSD
(PerkinElmer), and appropriate concentrations of test
compounds were added to 0.25 mL of 50 mM Tris–HCl
(pH 7.4) buffer containing 0.1% ascorbic acid and 4 mM
CaCl2. Non-specific binding was determined using 10 lM
methiothepin. The incubations were performed for 60 min
at 27 ꢀC, and these were terminated by rapid filtra-
tion through filtermat A glass fiber filter presoaked in 0.5%
PEI.
Measurement of morphine withdrawal syndrome
Morphine withdrawal syndrome was induced by the injec-
tion of naloxone (5 mg ⁄ kg), which is a competitive antago-
nist of opioid receptor. Immediately after the naloxone
injection, mice were placed into the individual observation
cylinders (25 cm in diameter and 50 cm in high) and the
frequency of jumps of each mouse was observed for
30 min.
For 5-HT7 receptor binding, cell membrane from stable
CHO-K1 cell line expressing the human recombinant 5-
HT7 receptor (PerkinElmer), 3 nM [3H]LSD and appropriate
concentrations of test compounds were added to 0.25 mL
of 50 mM Tris–HCl (pH 7.4) buffer containing 10 mM
MgSO4 and 0.5 mM EDTA. The mixture was incubated for
120 min at 27 ꢀC, and the reaction was terminated by
rapid filtration through filtermat A glass fiber filter presoa-
ked in 0.3% PEI. Non-specific binding was determined
using 10 lM methiothepin.
Receptor binding studies: serotonin receptor
For 5-HT1A receptor binding, aliquots of frozen membrane
(10 lg ⁄ well) from stable CHO-K1 cell line expressing the
human recombinant 5-HT1A receptor (PerkinElmer Life and
Analytical Sciences, Waltham, MA, USA), 0.25 nM [3H]8-
OH-DPAT (PerkinElmer) and appropriate concentrations of
test compounds were added to 0.25 mL of 50 mM Tris–
HCl (pH 7.4) buffer containing 10 mM MgSO4, 0.5 mM
EDTA, and 0.1% ascorbic acid. Incubations were carried
out for 60 min at 27 ꢀC, and these were terminated by
rapid filtration using a microbeta filtermate-96 harvester
(PerkinElmer) through filtermat A glass fiber filter presoaked
in 0.3% polyethylenimine (PEI). The filter was washed with
ice cold 50 mM Tris–HCl buffer solution (pH 7.4), and was
then covered with MeltiLex, sealed in a sample bag, dried
in an oven. The radioactivity retained in the filter was finally
counted using MicroBeta Plus (Wallac, Finland). Non-spe-
cific binding was defined with 0.5 lM of methiothepin. The
binding affinity (IC50) of a compound was calculated by
computerized non-linear regression analysis (GraphPad
Prism Program, San Diego, CA, USA) using 7–8 varied
concentrations of the compound run in duplicate tubes.
Primary cortical cell culture
Cortical cell culture was prepared from embryo of the
C57BL ⁄ 6 mice gestational age of 15 days. The brain was
dissected and kept in an ice-cold solution. The cortical tis-
sues were dissociated to single cells by a gentle suspen-
sion. The cell suspension was centrifuged at 112 · g for
5 min, and the resulting pellets were suspended in the
medium, MEM (Gibco, BRL, Rockville, MD, USA) supple-
mented with 5% heat-inactivated fetal calf serum and
house serum (Gibco), described on the plate onto coated
with Poly-D-Lysine (Sigma, St Louis, MO, USA). The cells
were cultured in a CO2 incubator (5% [v ⁄ v], 37 ꢀC). Seven
Chem Biol Drug Des 2013; 81: 389–398
393

Jung et al.
days after plating, the cells were treated with 10 lM cyto-
sine arabinofuranoside (Ara C) to reduce the growth of
contaminating non-neuronal cells. The cortical neuronal
cells were cultured in MEM supplemented with 10% horse
serum, but without glutamine.
5-trimethoxycinnamyl ketone (8) in 27% or 63% yield,
respectively (Scheme 1).
Treatment of acid 3 with 1.2 equivalents of thionyl chloride
afforded 3,4,5-trimethoxycinnamic chloride (5), which was
subsequently coupled with phenol to generate (E)-phenyl
3,4,5-trimethoxycinnamate (11) in 69% yield. Compound 3
was directly condensed with methanol or ethanol to give
(E)-methyl 3,4,5-trimethoxycinnamate (9) or (E)-ethyl 3,4,5-
trimethoxycinnamate (10) in 66% or 85% yield, respec-
tively. Indeed, (E)-benzyl 3,4,5-trimethoxycinnamate (12)
was obtained from acid 3 with treatment of 1.2 equivalents
of sodium bicarbonate and 1.2 equivalents of benzyl bro-
mide in 94% yield.
Western immunoblot analysis
(+)8-OH-DPAT (1 lM) was treated for various times (2, 5,
10, 30, 60 min) or was co-treated with WAY 100635
(1 lM), a selective antagonist of 5-HT1A receptor, for
30 min in cultured cortical neuronal cell. TMCA analogues
(10 lM) were treated with WAY 100635 (1 lM) for 30 min
in cortical neuronal cells. The cortical neuronal samples
were extracted with lysis buffer (150 mM NaCl, 50 mM
Tris–Cl pH 8, 1 mM EDTA, 1% Triton-X 100, 1 mM
PMSF, 1 mM Na3Vo4) and centrifuged at 16 300 · g for
15 min at 4 ꢀC. Protein concentrations were determined
using the protein assay kit (PIERCE Biotechnology, Rock-
ford, IL, USA). 4· sample buffer was added to extract
and boil for 4 min at 100 ꢀC. Equal amount of cellular
protein was separated by 10% polyacrylamide gels and
was electrophoretically transferred to polyvinylidene fluo-
ride (PVDF) membranes. Transfer blots were blocked in
3% skim milk in TBS-Tween 0.1% for 1 h and incubated
with antibodies specific to ERK1 ⁄ 2 and pERK1 ⁄ 2
(ERK1 ⁄ 2, 1:1000; and pERK1 ⁄ 2, 1:1000; Santa Cruz
Biotechnology, Santa Cruz, CA, USA), GAPDH (1:1000;
Cell signaling) at 4 ꢀC overnight. Blots were washed three
times for 30 min in 0.1% TBST, and incubated with an
appropriate horseradish peroxidase-conjugated secondary
antibody (Zymed Laboratiories, San Francisco, CA, USA).
Bound antibodies were visualized following chemilumines-
cence detection (GE Healthcare Life Science, Pittsburg,
PA, USA).
On the other hand, compound 3 was consequently cou-
pled with various amines [pyrrolidine, tetrahydrofurfuryl
amine, furfuryl amine, morpholine, 4-(3,5-dimethylphe-
nyl)piperazine, 4-benzylpiperidine, 4-ethylaniline] in the
presence of EDCI and HOBt in dichloromethane to give
amides 13–19 in 65%–93% yields, respectively.
Biology
Effects of TMCA derivatives on naloxone-induced
jumping behavior in morphine-dependent mice
The mice received morphine (10 mg ⁄ kg, i.p.) daily for
7 days to develop dependence on morphine and mor-
phine-induced withdrawal state (jumping) was examined
with TMCA analogues administration (20 mg ⁄ kg, i.p.). The
naloxone-induced jumping frequency was decreased by
treatment with TMCA analogues (Figure 2). Most of TMCA
derivatives decreased the jumping frequency in morphine-
dependent mice. Especially, TMCA analogues 14, 16, 17,
and 18 remarkably attenuated (about 90–98%) the nalox-
one-induced jumping frequency in morphine-dependent
mice. As expected (+)8-OH-DPAT, which is known as a 5-
HT1A receptor agonist, strongly suppressed the jumping
frequency (86%) in morphine-dependent mice. Of these
TMCA analogues, morpholin moiety 16 exhibited potent
inhibitory action to jumping frequencies which is indicator
of morphine-induced withdrawal symptom, while com-
pound 19 had not shown inhibitory action on jumping fre-
quencies compared with other TMCA analogues. In
addition, we found that the 1,4-diatomic moieties 14, 16,
17, and 18 showed excellent activities on naloxone-
induced jumping behavior in morphine-dependent mice.
Statistical analysis
All values are expressed as mean standard error (SE).
The results were subjected to an analysis of the variance
(ANOVA) using the Newman–Keuls Multiple Comparison
Test to analyze the difference.
Results and Discussion
Chemistry
A series of TMCA derivatives was prepared from commer-
cially available TMCA (3) in high yield. The generation of
ketones 7–8 was accomplished with 1.2 equivalents of
N,O-dimethylhydroxyamine via global coupling method as
EDCI and HOBt or 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluroniumhexafluorophosphate (HATU) ⁄ CH2Cl2 to
produce key intermediate N-methoxy-N-methyl 3,4,5-tri-
methoxycinnamamide (6), which was smoothly condensed
with freshly prepared Grignard Reagents (ethyl magnesium
bromide or phenyl magnesium bromide) to give (E)-ethyl
3,4,5-trimethoxycinnamyl ketone (7) and (E)-phenyl 3,4,
Receptor binding assay of TMCA derivatives
It was examined to identify how to show the biological
activity of TMCA derivatives in morphine withdrawal syn-
drome and in the receptor binding assay on serotonergic
(5-HT1A, 5-HT2A, 5-HT2C, 5-HT6, and 5-HT7). Interestingly,
all of tested TMCA derivatives showed high binding affinity
to the 5-HT1A receptor. The basal form of TMCA has a
high affinity to 5-HT1A and 5-HT2c receptor (Table 1).
394
Chem Biol Drug Des 2013; 81: 389–398

3,4,5-Trimethoxycinnamic Acid Derivatives
O
O
MeO
MeO
MeO
Cl
OH
a
MeO
OMe
5
OMe
3
d
e
c
O
O
Scheme 1: Synthesis of ketones
7–8, esters 9–12, and amides
13–19 from TMCA (3). Reagents
and conditions: (a) SOCl2 (1.2
equiv), reflux, 1 h; (b) various
amines, HOBt, EDCI, TEA,
CH2Cl2, rt, 24 h, 65–93%; (c)
N,O-dimethylhydroxylamine,
HOBt, EDCI, TEA, CH2Cl2, rt,
24 h (for 6, 75%) or
HATU ⁄ CH2Cl2, rt, 1 h, 80%; (d)
MeOH or EtOH, H2SO4 (cat) (for
9, 66%; for 10, 85%) or BnBr,
NaHCO2 (1.2 equiv), DMF ⁄ 1,4-
dioxane (1:1), 90 ꢀC, 24 h (for 12,
94%); (e) Phenol, 1 h, 69%; (f)
Et-MgBr or Ph-MgBr, THF,
)78 ꢀC fi rt, 2.5 h (for 7, 27%;
for 8, 63%).
MeO
MeO
OMe
MeO
N
R
b
f
Me
MeO
OMe
OMe
6
7–12
7
8
9
R = Et (27%)
R = Ph (63%)
R = OMe (66%)
10 R = OEt (85%)
11 R = OPh (69%)
12 R = OBn (94%)
O
13 R = Pyrrolidinyl (65%)
MeO
MeO
R
14 R = Tetrahydrofurfuryl (75%)
15 R = Furfuryl (81%)
16 R = Morpholinyl (85%)
17 R = 4-(3,5-Dimethoxy-phenyl)piperazinyl (93%)
18 R = 4-Benzylpiperidinyl (82%)
19 R = N-4-Ethylphenyl (80%)
OMe
13–19
(+)8-OH-DPAT induced elevation of extracellular
signal-regulated kinase1 ⁄ 2 (ERK1 ⁄ 2) expression in
cultured cortical neurons
We examined 5-HT1A receptor activation by measuring the
agonist-induced increase in pERK expression level in cul-
tured cortical neurons. As shown in Figure 3A, levels of
pERK were increased at 2, 5, 10, 30 min after treatment
with (+)8-OH-DPAT (1 lM) in cultured cortical neurons,
and pERK level was decreased to the basal level 60 min
after treatment with (+)8-OH-DPAT. The pERK levels were
highest at 30 min after treatment with (+)8-OH-DPAT, and
its elevation was suppressed by the 5-HT1A receptor-spe-
cific antagonist WAY 100635 (N-{2-[4-(2-methoxyphenyl)-
1-piperazinyl]
ethyl}-N-(2-pyridinyl)cyclohexanecarboxa-
mide, 1 lM). However, WAY 100635 alone did not affect
the level of pERK expression (Figure 3B).
Figure 2: Effects of TMCA derivatives 6–19 on naloxone-induced
jumping behavior in morphine-dependent mice. TMCA derivatives
(20 mg ⁄ kg, i.p.) and (+)8-OH-DPAT (0.1 mg ⁄ kg, i.p.) were injected
30 min prior to the morphine injection (10 mg ⁄ kg, i.p.). All the
mice were injected with the morphine or ⁄ and TMCA derivatives
for 7 days. On last day, naloxone (5 mg ⁄ kg, i.p.) was injected 6 h
after the final drug administration. Jumping frequency was
observed for 30 min in transparent cylinder. Inhibition of jumping
frequency (%) is compared with morphine-treated group.
#p < 0.01 in comparison with control, *p < 0.05, **p < 0.01 in
comparison with morphine.
Effect of 5-HT1A receptor-specific antagonist on
TMCA derivatives-induced pERK expression in
cultured cortical neurons
We determined all of new synthesized TMCA derivatives on
5-HT1A receptor activities. We confirmed that the effect of
TMCA derivatives on activation of ERK1 ⁄ 2 and its possible
action as the 5-HT1A receptor agonist using WAY 100635,
a selective 5-HT1A receptor antagonist. As shown in Fig-
Chem Biol Drug Des 2013; 81: 389–398
395

Jung et al.
Table 1: Receptor binding affinity of TMCA derivatives
Receptor binding affinity (IC50, lM)a
mice. Interestingly, most of our synthesized TMCA deriva-
tives effectively decreased jumping frequencies, which is an
indication of grade of morphine withdrawal syndrome. In
agreement with earlier findings (20), the treatment of (+)8-
OH-DPAT attenuated the naloxone-induced jumping
behavior in morphine-dependent mice. It was known that
5-HT selectively inhibits excitatory amino acid input to the
LC during opiate withdrawal (19). Several selective 5-HT1A
agonists include (+)8-OH-DPAT that attenuate the antinoci-
ception in l-opioid agonist morphine, not j-opioid agonist
(22). In our results, synthesized TMCA derivatives have high
affinity on 5-HT1A receptor. Our results suggest that the
TMCA derivatives show antinarcotic effect possibly via acti-
vation of 5-HT1A receptors. Using primary cortical neuronal
cells expressing 5-HT1A receptors as a model system, we
confirmed that stimulation of the 5-HT1A receptor was
determined by the measurement of ERK1 ⁄ 2 expression.
We observed the effect of 5-HT1A receptors agonist, (+)8-
OH-DPAT, on activation ERK1 ⁄ 2 in our cellular system.
Synthesized TMCA derivatives induced elevation of ERK1 ⁄ 2
level and its elevation was blocked by the WAY 100635. It
might be suggested that the role of antinarcotics effect of
synthetic TMCA derivatives might be due to the agonistic
action on 5-HT1A receptor in morphine dependent mice.
Compound
5-HT1A
5-HT2A
5-HT2C
5-HT6
5-HT7
6
7
8
9
2.7
7.4
9.4
4.1
4.8
1.1
2.4
1.3
1.1
4.5
8.5
1.5
2.0
2.5
7.6
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.5
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
8.8
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
10
11
12
13
14
15
16
17
18
19
TMCA
2.5
>10
TMCA, 3,4,5-trimethoxycinnamic acid.
aAll compounds were tested at maximum 10 lM of concentration,
and their binding affinities were calculated to IC50 (lM).
ure 4, when the cells were treated (10 lM) with TMCA
derivatives 6, 8, 11, 15, and 18 for 30 min, derivatives stim-
ulated the activation of ERK1 ⁄ 2 expression in cortical neu-
ronal cells. However, other TMCA derivatives did not
strongly stimulate the activation of ERK1 ⁄ 2 (Data not
shown). The elevation of pERK expression by TMCA deriv-
atives 6, 8, 11, 15, and 18 treatment was suppressed by
WAY 100635 (1 lM), 5-HT1A receptor-specific antagonist.
Conclusion
A series of TMCA derivatives 6–19 was synthesized to
elucidate properties of an antinarcotic effect on morphine
dependence in mice and their binding affinities on dopa-
minergic and serotonergic receptors. The synthetic
approach involves condensation and coupling reaction of
acid 3. We found that most of TMCA derivatives
(20 mg ⁄ kg ⁄ day) have high affinity to 5-HT1A receptor as
well as strong suppressing effect on the naloxone-
We here examined the role of new synthetic TMCA deriva-
tives as antinarcotics, especially in morphine. Administration
of the 5-HT1A agonist, (+)8-OH-DPAT, inhibited naloxone-
induced withdrawal syndrome in morphine-dependent
A
B
Figure 3: Elevation of pERK level by (+)8-OH-DPAT treatment in cultured cortical neurons. Cells were treated with (+)8-OH-DPAT and
each cell was taken at variable time (2, 5, 10, 30, 60 min) (A). Cells were treated with (+)8-OH-DPAT (1 lM) and ⁄ or WAY 100635 (1 lM)
for 30 min (B). The expression of pERK level was examined using Western blot analysis.
Figure 4: The effects of WAY 100635 on the TMCA derivatives-induced elevation of pERK levels. Cultured neuronal cells were treated
with TMCA derivatives (10 lM) and ⁄ or WAY 100635 (1 lM) for 30 min. The expression of pERK levels was examined using Western blot
analysis.
396
Chem Biol Drug Des 2013; 81: 389–398

3,4,5-Trimethoxycinnamic Acid Derivatives
induced jumping behavior in morphine-dependent mice. It
was found that the compound 16 exhibited strong inhibi-
tory effect on the naloxone-induced jumping behavior in
morphine-dependent mice among these analogues. In
terms of structural characteristics, the presence of a mor-
pholine group and a piperazine group at amide parts
was important functionally for an antinarcotic efficacy and
binding affinity. We have expected that simple synthesis
of new TMCA derivatives and key fragments are useful
for the modification of antinarcotic effect on morphine
dependence in mice.
9. Kawashima K., Miyako D., Ishino Y., Makino T., Saito K.,
Kano Y. (2004) Anti-stress effects of 3,4,5-trimethoxy-
cinnamic acid, an active constituent of roots of Polygala
tenuifolia (Onji). Biol Pharm Bull;27:1317–1319.
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Acknowledgments
12. Glod L.H., Stinus L., Inturrisi C.E., Koob G.F. (1994)
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368–371.
This study was supported by the Korea Research Founda-
tion Grant (MRC, 2010-0029355) funded by the Korean
Government (MEST) and supported by Basic Science
Research Program through the National Research Founda-
tion of Korea (NRF) funded by the Ministry of Education,
Science and Technology (2012-0007574).
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