Total synthesis and antioxidative activity of magnolamide from Magnolia coco

Article abstract of DOI:10.3987/COM-05-10344

A simple total synthesis of naturally occurring magnolamide, N-[4-(2-formyl-5-hydroxymethylpyrrol-1-yl)butyl]-3-(4-hydroxy-3-methoxyphenyl) -2-propenamide, from pyrrole-2,5-dicarbaldehyde was performed in six steps and its antioxidant activity was compare

Full text of DOI:10.3987/COM-05-10344

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HETEROCYCLES, Vol. 65, No. 5, 2005, pp. 1215 - 1220
Received, 20th January, 2005, Accepted, 24th February, 2005, Published online, 1st March, 2005
TOTAL SYNTHESIS AND ANTIOXIDATIVE ACTIVITY OF
MAGNOLAMIDE FROM MAGNOLIA COCO
Wen-Fei Chiou,a Chien-Chang Shen,a Hsi-Jung Yu,b Chang-Han Chiang,b
Ching-Chuan Chen,b Wenyi Chang,a and Ming-Jaw Dona*
aNational Research Institute of Chinese Medicine, 155-1, Section 2, Li-Nung
Street, Taipei 112, Taiwan, Republic of China
E-mail: mjdon@nricm.edu.tw
bDepartment of Chemistry, Chinese Culture University, 55, Hwa-Kang Road,
Yang-Ming-Shan, Taipei 111, Taiwan, Republic of China
Abstract – A simple total synthesis of naturally occurring magnolamide,
N-[4-(2-formyl-5-hydroxymethylpyrrol-1-yl)butyl]-3-(4-hydroxy-3-methoxy-
phenyl)-2-propenamide, from pyrrole-2,5-dicarbaldehyde was performed in six
steps and its antioxidant activity was compared with probucol and resveratrol.
Results showed that magnolamide markedly inhibited CuSO4-induced oxidation
of human low-density lipoprotein (IC50 = 9.7 µM) with similar activity to
probucol and resveratrol.
INTRODUCTION
Magnolamide, a phenolic amide, was isolated and characterized from the methanolic extract of the leaves
of Magnolia coco (Magnoliaceae).1 The stems and leaves of M. coco are used as a Chinese herbal
medicine against liver disease. Magnolamide was the first naturally occurring amide that has a
[(2-formyl-5-hydroxymethyl)pyrrolyl]butylamine moiety as an amine unit, and its biological activity has
not been reported yet. According to the literature reports, hydroxylated and/or methoxylated cinnamic
acids conjugated with different kinds of amine (putrescine,2 spermidine,2 spermine,2 tyramine,2-5
serotonine6,7) were found from various plants. A number of amides are known to have biological
activities such as inhibitory effects against platelet aggregation,5 antioxidative activity,6,8 and inhibition of
in vitro prostaglandin biosynthesizing enzyme (PG synthetase).9 In order to obtain more material for
further study of the biological activities, we were prompted to investigate the synthesis and antioxidative
activity of magnolamide. Herein, we wish to report a facile preparation of magnolamide, the structure of

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which was established by spectroscopic analysis and by comparison with published data of the natural
product. The inhibitory effect of magnolamide on CuSO4-induced human low-density lipoprotein (LDL)
oxidation was also discussed.
RESULTS AND DISCUSSION
The synthetic strategy shown in Scheme 1 was employed for the preparation of magnolamide. The
starting pyrrole-2,5-dicarbaldehyde was prepared from pyrrole-2-carbaldehyde by a known literature
procedure.10 N-Alkylation of pyrrole-2,5-dicarbaldehyde with N-(4-bromobutyl)phthalimide in the
presence of K2CO3 in acetonitrile gave pyrrole derivative (1). Two formyl groups on pyrrole ring of 1
were protected with 1,3-propanedithiol in the presence of anilinium chloride in MeOH to yield
1,3-dithiane derivative (2), which was then hydrolyzed with hydrazine hydrate to afford the amine
derivative (3) in good yield. Condensation of 3 with 4-hydroxy-3-methoxycinnamic acid in the presence
of benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) and triethylamine
in DMF gave amide (4).8 Treatment of 4 with HgO and HgCl2 to remove the dithiane protection
regenerated the formyl group on pyrrole ring to give 5, which was reduced to magnolamide (6) using
NaBH4 in 92% yield.
S
CHO
CHO
NH
O
O
S
i
iii
ii
N
N
N
N
OHC
OHC
S
S
O
O
1
2
S
S
O
S
S
iv
v
MeO
HO
N
S
N
N
NH2
H
S
S
S
4
3
OHC
OHC
O
O
vi
MeO
HO
N
MeO
HO
N
N
N
H
H
CHO
OH
5
6, magnolamide
Scheme1. Reagents and conditions: (i) N-(4-bromobutyl)phthalimide, K2CO3, MeCN, reflux; (ii)
1,3-propanedithiol, anilinium chloride, MeOH; (iii) hydrazine hydrate, THF, reflux; (iv)
4-hydroxy-3-methoxycinnamic acid, Et3N, BOP, CH2Cl2; (v) HgCl2, HgO, MeCN/H2O; (vi) NaBH4,
MeOH.
The antioxidative activities of magnolamide (6) and its analogue (5) were evaluated in the inhibitory
effects of CuSO4-induced LDL lipid oxidation, and results are shown in Table 1. Both compounds

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inhibited conjugated diene formation in a concentration-dependent manner with IC50 values of 9.7 and
16.9 µM, respectively. Resveratrol and probucol were used as two reference antioxidants. Results showed
that the antioxidative activity of magnolamide was similar to that of probucol and resveratrol.
Table 1. The IC50 value of 5 and 6 in the inhibition of Cu2+-induced LDL lipid oxidation.
5
6
resveratrol
probucol
IC50 (µM)
16.9±2.3
9.7±2.8
13.1±2.6
8.7±1.4
In conclusion, we have presented here a simple total synthesis of magnolamide in six steps from
pyrrole-2,5-dicarbaldehyde with an overall yield of 34%. Spectroscopic data of the synthesized product
were identical with those of the natural product (6).1 Magnolamide showed significant inhibitory effect in
Cu2+/O2-induced LDL lipid peroxidation. Synthesis of other analogues of magnolamide is in progress
with an aim to further improve the antioxadative activity.
EXPERIMENTAL
Melting points were determined with a Yanaco micromelting point apparatus and are uncorrected. IR
spectra were obtained on a Nicolet Avatar 320 FTIR spectrophotometer. NMR spectra were recorded on a
Varian Unity Inova-500 and Gemini-200 spectrometers. Chemical shifts are reported in parts per million
(δ) units relative to internal tetramethylsilane. The EIMS spectra were measured from a Finnigan GCQ
GC/MS spectrometer at 30 eV. HREIMS was recorded on a Finnigan MAT 95S mass spectrometer.
Elemental analyses were performed on Perkin-Elmer CHN-2400 analyzer. Column chromatography was
performed with E. Merck 230-400 mesh silica gel.
1-[4-(1,3-Dioxoisoindolin-2-yl)butyl]-1H-pyrrole-2,5-dicarbaldehyde (1). To
a
solution of
pyrrole-2,5-dicabaldehyde (1.58 g, 12.8 mmol) in acetonitrile (60 mL) were added
N-(4-bromobutyl)phthalimide (4.0 g, 14.2 mmol) and K2CO3 (3.0 g, 21.7 mmol), and the mixture was
refluxed for overnight. After cooling, the solution was filtered and evaporated. The residue was taken in
chloroform (100 mL) and washed with water (100 mL). The aqueous layer was extracted once again with
CHCl3 (100 mL). The combined organic extracts were washed with saturated aqueous NaCl, dried over
Na2SO4, and evaporated. The residue was recrystallized from CHCl3/hexane to give 1 (3.32 g, 80 %) as a
1
white solid: mp 195-197 °C; IR (KBr) 2938, 1767, 1717, 1670, 1397, 1199, 1051, 724 cm-1; H NMR
(CDCl3, 500 MHz) δ 1.66-1.72 (m, 2H), 1.76-1.82 (m, 2H), 3.67 (t, J = 7.0 Hz, 2H), 4.78 (t, J = 7.0 Hz,
2H), 6.93 (s, 2H), 7.67-7.70 (m, 2H), 7.79-7.82 (m, 2H), 9.78 (s, 2H); EIMS m/z (%) 324 (57) [M+], 295
(68), 148 (96), 136 (100), 120 (56); Anal. Calcd for C18H16N2O4: C, 66.66; H, 4.97; N, 8.64. Found: C,
66.43; H, 4.94; N, 8.58.
2-{4-[2,5-Di(1,3-dithian-2-yl)-1H-pyrrol-1-yl]butyl}isoindoline-1,3-dione (2). To a solution of 1 (3.30

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g, 10.2 mmol) in MeOH (100 mL) were added 1,3-propanedithiol (2.32 g, 21.4 mmol) and anilinium
chloride (70 mg), and the mixture was stirred at rt for 3 h. The solution was then filtered and evaporated.
The residue was taken in chloroform (100 mL) and washed with water (100 mL). The organic layer was
dried over Na2SO4, and evaporated. The residue was recrystallized from CHCl3/hexane to give 2 (4.83 g,
1
94 %) as a white solid: mp 183-185 °C; IR (KBr) 1772, 1717, 1396, 1363, 1036, 721 cm-1; H NMR
(CDCl3, 500 MHz) δ 1.78-1.91 (m, 6H), 2.08-2.13 (m, 2H), 2.77-2.81 (m, 4H), 3.01-3.06 (m, 4H), 3.76 (t,
J = 6.5 Hz, 2H), 4.06 (t, J = 7.0 Hz, 2H), 5.15 (s, 2H), 6.24 (s, 2H), 7.68-7.71 (m, 2H), 7.80-7.83 (m,
2H); EIMS m/z (%) 504 (100) [M+], 430 (30), 398 (42), 385 (26), 324 (16), 119 (18); Anal. Calcd for
C24H28N2O2S4: C, 57.11; H, 5.59; N, 5.55; S, 25.41. Found: C, 56.85; H, 5.71; N, 5.47; S, 25.54.
4-[2,5-Di(1,3-dithian-2-yl)-1H-pyrrol-1-yl]butyl-1-amine (3). To a solution of 2 (2.50 g, 4.96 mmol) in
THF (100 mL) was added hydrazine hydrate (0.6 g, 12.0 mmol), and the mixture was refluxed for
overnight. After the mixture was cooled, the pH of the mixture was adjusted to 4.0 with 1N HCl, and the
precipitate was then filtered. The filtrate was evaporated to small amount under reduced pressure and
adjusted pH to 9.0 with 2N NaOH solution. The resulting solution was extracted with chloroform three
times. The combined extracts were washed with water, dried over Na2SO4, and concentrated. The residue
was recrystallized from CHCl3/hexane to give 3 (1.60 g, 86 %) as a white solid, which was used in the
next step without further purification: mp 121-122 °C; 1H NMR (CDCl3, 200 MHz) δ 1.49-1.60 (m, 2H),
1.80-2.17(m, 6H), 2.73-3.09 (m, 10H), 4.03 (t, J = 8.0 Hz, 2H), 5.16 (s, 2H), 6.26 (s, 2H); EIMS m/z (%)
374 (25) [M+], 255 (100), 119 (72).
(E)-N-{4-[2,5-Di(1,3-dithian-2-yl)-1H-pyrrol-1-yl]butyl}-3-(4-hydroxy-3-methoxyphenyl)-2-propen-
amide (4). To a stirred solution of 4-hydroxy-3-methoxycinnamic acid (388 mg, 2.0 mmol) in DMF (8
mL) and Et3N (0.28 mL) cooled to 0 °C were added 3 (860 mg, 2.3 mmol) and a solution of BOP (884
mg, 2.0 mmol) in CH2Cl2 (8 mL). The mixture was stirred at 0 °C for 30 min and then at rt for 2 h. The
solution was poured into a mixture of H2O (100 mL) and CHCl3 (100 mL). The layer was separated, and
the aqueous phase was extracted with CHCl3. The combined organic extracts were washed successive
with 1 N HCl, water, 1N NaHCO3, water, dried over Na2SO4, filtered, and evaporated. The residue was
purified by silica gel chromatography eluting with 5% acetone in CHCl3 to give 4 as a white solid (890
mg, 81%): mp 192-194 °C (recrystallized from CHCl3); IR (KBr) 3302, 2935, 1688, 1658, 1513, 1272,
1183, 816 cm-1; 1H NMR (CDCl3, 200 MHz) δ 1.61-2.15 (m, 8H), 2.77-3.09 (m, 8H), 3.42 (q, J = 6.2 Hz,
2H), 3.89 (s, 3H), 4.07 (t, J = 7.6 Hz, 2H), 5.15 (s, 2H), 5.84 (br t, 1H, NH), 6.20 (d, J = 15.6 Hz, 1H),
6.28 (s, 2H), 6.88 (d, J = 8.4 Hz, 1H), 6.95 (d, J = 1.6 Hz, 1H), 7.07 (dd, J = 8.4, 1.6 Hz, 1H), 7.50 (d, J =
15.6 Hz, 1H); EIMS m/z (%) 550 (40) [M+], 431 (100), 251 (97), 177 (63); Anal. Calcd for
C26H34N2O3S4: C, 56.69; H, 6.22; N, 5.09; S 23.29. Found: C, 56.46; H, 6.16; N, 5.09; S, 23.12.
(E)-N-[4-(2,5-Diformyl-1H-pyrrol-1-yl)butyl]-3-(4-hydroxy-3-methoxyphenyl)-2-propenamide (5).

HETEROCYCLES, Vol. 65, No. 5, 2005
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To a solution of 4 (750 mg, 1.36 mmol) in acetonitrile/H2O (85:15, 50 mL) were added HgO (1.20 g, 5.44
mmol) and HgCl2 (1.50 g, 5.44 mmol), and the mixture was stirred at rt for 2 h. The solution was then
filtered and evaporated. The residue was purified by silica gel chromatography eluting with 3% methanol
in CH2Cl2 to give 5 as a white solid (360 mg, 71%): mp 141-143 °C (recrystallized from methanol); IR
1
(KBr) 3317, 2954, 2930, 1650, 1619, 1548, 1513, 1269, 1218, 1125, 971, 790 cm-1; H NMR (CDCl3,
500 MHz) δ 1.61-1.65 (m, 2H), 1.75-1.80 (m, 2H), 3.43 (t, J = 6.5 Hz, 2H), 3.87 (s, 3H), 4.73 (t, J = 7.5
Hz, 2H), 6.01 (s, 1H, OH), 6.24 (br t, 1H, NH), 6.27 (d, J = 15.5 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 6.95 (s,
2H), 6.95 (d, J =1.5 Hz, 1H), 7.03 (dd, J = 8.5, 1.5 Hz, 1H), 7.50 (d, J = 15.5 Hz, 1H), 9.79 (s, 2H); EIMS
m/z (%) 370 (100) [M+], 177 (61), 165 (60), 149 (99), 135 (73), 121 (40); Anal. Calcd for C20H22N2O5•1/2
CH3OH: C, 63.73; H, 6.22; N, 7.25. Found: C, 63.69; H, 6.54; N, 6.95.
(E)-N-[4-(2-Formyl-5-hydroxymethyl-1H-pyrrol-1-yl)butyl]-3-(4-hydroxy-3-methoxyphenyl)-2-pro-
enamide (Magnolamide, 6). To a solution of 5 (290 mg, 0.78 mmol) in MeOH (20 mL) was added
NaBH4 (14.4 mg, 0.39 mmol), and the mixture was stirred at rt for 15 min. The solution was then
evaporated, and the residue was purified by silica gel chromatography eluting with 5% acetone in CHCl3
1
to give 6 (267 mg, 92%, hygroscopic) as a colorless oil: IR (neat) 3419, 1650, 1515, 1272, 773 cm-1; H
NMR (CD3OD, 500 MHz) δ 1.56-1.62 (m, 2H), 1.76-1.82 (m, 2H), 3.31 (t, J = 7.0 Hz, 2H), 3.86 (s, 3H),
4.38 (t, J = 7.5 Hz, 2H), 4.62 (s, 2H), 6.24 (d, J = 4.0 Hz, 1H), 6.39 (d, J =15.5 Hz, 1H), 6.77 (d, J = 8.5
Hz, 1H), 6.96 (d, J = 4.0 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 7.09 (s, 1H), 7.42 (d, J = 15.5 Hz, 1H), 9.40 (s,
1H); 13C NMR (CD3OD, 125 MHz) δ 27.7, 29.8, 40.0, 46.3, 56.3, 56.5, 111.5, 116.7, 118.3, 123.4, 126.4,
127.7, 133.5, 142.2, 144.5, 149.6, 150.8, 169.3, 180.9; EIMS m/z (%) 372 (95) [M+], 343 (99), 195 (19),
177 (100), 145 (30); HR-EIMS m/z 372.1694 (calcd for C20H24N2O3, 372.1680).
Assay of LDL lipid peroxidation
LDL (d = 1.019-1.063) was isolated from freshly prepared human plasma and stored in the dark at 4 °C
under nitrogen for up to 2 weeks. LDL concentration was determined by using Biorad protein assay kit
(USA) and the purity was assessed by agarose gel electrophoresis (Paragen, Beckman LIPO kit).
Aliquots of LDL (50 µg/mL) was incubated with copper sulphate (10 µM final concentration) at 37 °C for
6 h, by monitoring every 15 min the absorbance at 234 nm of conjugated dienes.11 Oxidations were
performed in the presence or absence of tested drugs concentrations ranging from 1 to 25 µM. The IC50
was calculated as the 50 % inhibition of peak absorbance at 234 nm.
ACKNOWLEDGEMENTS
This research was supported by grants from the National Science Council of Republic of China (NSC
91-2113-M-077-003).

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