Total synthesis and the biological activities of (±)- norannuradhapurine

Article abstract of DOI:10.3390/molecules14010089

The structure previously assigned to the phenolic noraporphine alkaloid, (-)-norannuradhapurine has been confirmed by a total synthesis of the racemic alkaloid in which the key step involved the formation of the C ring by a radical-initiated cyclization.

Full text of DOI:10.3390/molecules14010089

Molecules 2009, 14, 89-101; doi:10.3390/molecules14010089
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Total Synthesis and the Biological Activities of
(±)-Norannuradhapurine
Surachai Nimgirawath 1,*, Rujee Lorpitthaya 1, Asawin Wanbanjob 1, Thongchai Taechowisan 2
and Yue-Mao Shen 3
1
Department of Chemistry, Faculty of Science, Silpakorn University, Nakorn Pathom 73000,
Thailand; E-mails: G050010@ntu.edu.sg (R. L.), winsusc@gmail.com (A. W.)
2
Department of Microbiology, Faculty of Science, Silpakorn University, Nakorn Pathom 73000,
Thailand; E-mail: tthongch@su.ac.th (T. T.)
3
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, P. R. China. E-mail:
yshen@mail.kib.ac.cn (Y-M. C.)
* Author to whom correspondence should be addressed; E-mail: surachai@su.ac.th.
Received: 2 December 2008; in revised form; 19 December 2008 / Accepted: 23 December 2008 /
Published: 29 December 2008
Abstract: The structure previously assigned to the phenolic noraporphine alkaloid,
(-)-norannuradhapurine has been confirmed by a total synthesis of the racemic alkaloid in
which the key step involved the formation of the C ring by a radical-initiated cyclization.
although inactive against Staphylococcus aureus ATCC25932, Escherichia coli
ATCC10536 and Candida albicans ATCC90028, (±)-norannuradhapurine inhibits the
production of NO, PGE2, TNF-α, IL-1β and IL-6 and the expression of iNOS and COX-2
in RAW 264.7 macrophages stimulated with LPS in vitro.
Keywords: Alkaloid; Anti-inflammatory Activity; Aporphine; Isoquinoline; Synthesis;
Radical Cyclization.

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Introduction
(-)-Norannuradhapurine (1a) is a phenolic noraporphine alkaloid isolated for the first time from the
bark and leaves of Polyalthia acuminate Thw. (Annonaceae) [1] and subsequently from the bark and
wood of Fissistigma glaucescens (Hance) Merr. and the wood of Fissistigma oldhamii (Hemsl.) Merr.
(Annonaceae) [2]. (-)-Norannuradhapurine has been shown to exhibit strong inhibition of adenosine
5’-diphosphate (ADP)-induced, collagen-induced and platelet-activating factor (1-O-alkyl-2-acetyl-
sn-glycero-3-phosphocholine)-induced platelet aggregations [3]. In addition, (-)-norannuradhapurine
has exhibited a broad spectrum of growth inhibitory activities against murine and human leukemic
cells with IC50 values around 3mM and it also has strong inhibitory effects on DNA, RNA and protein
biosynthesis [4]. In view of these interesting biological activities and the fact that the alkaloid occurs
only in minute quantities in Nature, we decided to undertake a total synthesis of the alkaloid to
establish its structure and also to make it more accessible for anti-microbial and anti-inflammatory
activity studies.
Results and Discussion
Synthesis
The strategy employed for the synthesis of (±)-norannuradhapurine (1a) was based on the
construction of ring C by a radical-initiated cyclization which has proved successful in the syntheses of
other aporphine alkaloids [5-8] (Scheme 1).
For this purpose, the required starting materials were 3,4-methylene-dioxyphenethylamine (2) [9]
and 2-benzyloxy-6-bromo-3-methoxyphenylacetic acid (3e). Thus, benzylation of 6-bromo-2-hydroxy-
3-methoxybenzaldehyde [10] gave 3a, which was converted into 3b-e, respectively by conventional
methods. Condensation of 2-benzyloxy-6-bromo-3-methoxy-phenylacetyl chloride (3f) with 3,4-
methylenedioxyphenethylamine (2) gave amide 4, which was converted into 5 by a Bischler-
Napieralski reaction. Reduction of 5 with sodium borohydride gave 6a which was treated with
trifluoroacetic anhydride to give 6b. Treatment of 6b with tributyltin hydride and 2,2′-
azobis(isobutyronitrile) afforded 1b in 40% yield. The structure of 1b was supported by 1H-NMR data,
in which H-11 gave rise to an unusually low-field doublet at δ 7.86 and the 1,2-methylenedioxy
protons gave rise to an AB quartet at δ 6.02 (J = 1.20 Hz ), characteristic of an aporphine moiety
bearing a methylenedioxy group at those positions. Attempts to remove the trifluoroacetyl group from
1b under basic conditions were fruitless, possibly due to steric hindrance of the benzyloxy group on
ring D. Fortunately, catalytic hydrogenolysis of 1b went smoothly to give 1c, whose trifluoroacetyl
group was smoothly removed to give (±)-norannuradhapurine (1a), the 1H-NMR spectral data of which
were in excellent agreement with those reported for natural (-)-norannuradhapurine [1]. Since the 13C-
NMR spectral data of the natural alkaloid have not been previously reported, we have reported here
these data from spectra measured both in CDCl3 and d6-DMSO for future reference.

Molecules 2009, 14
91
Scheme 1. Synthetic route to (±)-norannuradhapurine (1a).
R
O
O
O
B r
O B n
O C H
N
N H
2
R
1
O
3
2
3 a : R = C H O
A
3 b : R = C H O H
R
2
2
B
C
D
E
3 c : R = C H C l
3 d : R = C H 2 C N
O C H
3
2
1 b : R = C O C F 3 , R = O B n
3 e : R = C H C O O H
1
2
2
K
L
1 c : R = C O C F 3 , R = O H
3 f : R = C H C O C l
2
2
1 a : R 1 = H , R = O H
1
2
O
O
O
N
N H
N
O
H
O
F
O
G
O
R
J
B r
B r
1 b
B r
( 2 + 3 f )
O B n
O B n
O B n
O C H
O C H
3
3
O C H
3
6 a : R = H
6 b : R = C O C F
I
4
5
3
Reagents and conditions: A) NaBH4/ethanol; B) SOCl2, pyridine/chloroform; C) NaCN, KI/ water-
acetone; D) i. KOH/water-ethanol; ii. H3O+; E) SOCl2/benzene; F) 10% NaHCO3/ chloroform; G)
POCl3/benzene; H) NaBH4/ethanol; I) (CF3CO)2O, Et3N/chloroform; J) Bu3SnH, AIBN/dry
toluene; K) H2/Pd/C/ethanol; L) K2CO3/methanol-water.
Biological activity
At a concentration of 256 μg/mL, (±)-norannuradhapurine was inactive Staphylococcus aureus
ATCC25932 Escherichia coli ATCC10536 and Candida albicans ATCC90028. In the course of our
studies on its anti-inflammatory activity, we have found that (±)-norannuradhapurine inhibits NO
production in murine macrophage RAW 264.7 cells stimulated with LPS (Figure 1). Next we
investigated the effect of (±)-norannuradhapurine on the release of PGE2. Compared with the untreated
control, LPS (1 μg/mL) induced a great production of PGE2 in RAW 264.7 cells.
(±)-Norannuradhapurine (1–5 mg/mL) inhibited the production of PEG2 in RAW 264.7 cells
stimulated with LPS in a concentration-dependent manner (Figure 2). To elucidate the mechanism of
the inhibitory effect of (±)-norannuradhapurine on NO and PGE2 production, we investigated their
effects on iNOS and COX-2 expression levels, respectively. In response to LPS, the iNOS and COX-2
induction were markedly increased, (±)-norannuradhapurine significantly decreased the iNOS and
COX-2 protein expression in a concentration-dependent manner (Figures 3, 4).

Molecules 2009, 14
Figure 1. Evaluation of nitrite production by RAW 264.7 cells stimulated for 24 hours
92
with LPS alone or combination with increasing concentrations (1-5 μg/mL) of (±)-
norannuradhapurine. The values are the means of at least three determinations ± SD.
Probability levels (Student’s t-test): * p < 0.05 vs. LPS-treated group.
LPS 1μg/mL
70
60
50
*
40
30
*
20
10
*
0
1
2.5
5
blank
ctrl
[(±)-norannuradhapurine] ug/mL
Figure 2. Effect of (±)-norannuradhapurine on PEG2 production in LPS-induced RAW
264.7 macrophage for 24 hours. The values are the means of at least three determinations
± SD. Probability level(Student’s t-test): * p < 0.05 vs. LPS-treated group.
LPS 1μg/mL
35
30
25
20
*
15
10
5
*
0
1
2.5
5
blank
ctrl
[(±)-norannuradhapurine] ug/mL
Figure 3. Effect of (±)-norannuradhapurine on iNOS protein production by LPS-induced
RAW 264.7 macrophage for 24 hours.
LPS 1μg/mL
12
10
8
6
4
2
0
1
2.5
5
blank
Ctrl
(±)-norannuradhapurine

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93
Figure 4. Effect of (±)-norannuradhapurine and LPS-induced COX-2 protein expression in
RAW 264.7 cells.
LPS 1μg/mL
12
10
8
6
4
2
0
1
2.5
5
blank
Ctrl
(±)-norannuradhapurine
In contrast to iNOS and COX-2, (±)-norannuradhapurine had no effect on the expression of β-actin
and COX-1 (data not shown). This finding indicates that (±)-norannuradhapurine could suppress NO
and PGE2 production in LPS-stimulated RAW 264.7 cells by inhibiting iNOS and COX-2 protein
expression, respectively. It has been reported that cytokines such as TNF-α, IL-1β and IL-6 are pro-
inflammatory in vitro as well as in vivo [14]. The present study also demonstrated that (±)-
norannuradhapurine has inhibitory effects on the production of TNF-α, IL-1β and IL-6 in LPS-
stimulated RAW 264.7 cells. As shown in Figures 5, 6, 7, LPS-induced productions of TNF-α, IL-1β
and IL-6 were significantly inhibited by (±)-norannuradhapurine in a concentration-dependent manner.
In addition, the cytotoxic effect of (±)-norannuradhapurine was evaluated in the absence or presence of
LPS, (more than 95% cell viability). There is no significant difference on cell viability when treated
with (±)-norannuradhapurine at all concentrations used (1-5 μg/mL) in the absence or presence of LPS.
Figure 5. Effect of (±)-±)-norannuradhapurine on LPS-induced TNF-α production by
RAW 264.7 cells. The values are the means of at least three determinations ± SD.
Probability level (Student’s t-test): * p < 0.05 vs. LPS-treated group.
LPS 1μg/mL
40
35
30
*
25
*
20
15
10
*
5
0
1
2.5
5
blank
ctrl
[(±)-norannuradhapurine] ug/mL

Molecules 2009, 14
Figure 6. Effect of (±)-norannuradhapurine on IL1-β production by RAW 264.7 cells. The
94
values are the means of at least three determinations± SD. Probability level (Student’s t-
test): * p < 0.05 vs. LPS-treated group.
40
LPS 1μg/mL
35
30
*
25
20
*
15
10
5
0
1
2.5
5
blank
ctrl
(±)-norannuradhapurine] ug/mL
Figure 7. Effect of (±)-norannuradhapurine on LPS induced IL-6 production by RAW
264.7 cells. The values are the means of at least three determinations ± SD. Probability
level (Student’s t-test): * p < 0.05 vs. LPS-treated group.
LPS 1μg/mL
30
25
20
15
10
5
*
*
0
1
2.5
5
blank
ctrl
[(±)-norannuradhapurine] ug/mL
Conclusions
In conclusion, we found that (±)-norannuradhapurine has anti-inflammatory activity, it inhibits the
production of NO, PGE2, TNF-α, IL-1β and IL-6 and the expression of iNOS and COX-2 in
macrophages stimulated with LPS in vitro. From these results it is expected that (±)-
norannuradhapurine could be potentially useful for the treatment inflammatory diseases.

Molecules 2009, 14
95
Experimental
General
Melting points were determined on a Stuart SMP2 apparatus and are uncorrected. Ultraviolet
spectra were recorded on methanol solutions with a Jasco V-530 UV-VIS spectrophotometer. Infrared
spectra were recorded on Nujol mulls unless stated otherwise with a Perkin-Elmer Spectrum GX FT-
1
IR spectrophotometer. H- and 13C-NMR magnetic resonance spectra were recorded on CDCl3
1
13
solutions, unless stated otherwise, at 300 MHz for H and 75 MHz for C with a Bruker AVANCE
300 spectrometer. Tetramethylsilane was used as the internal standard. Mass spectra were measured on
a Hewlett Packard 5989B spectrometer. Elemental microanalyses were performed with a Perkin-Elmer
2400 elemental analyser.
2-Benzyloxy-6-bromo-3-methoxybenzaldehyde (3a). Using standard conditions, 3a was obtained in
1
92.4% yield as a green solid, m.p.53-54°C (Lit. [11] m.p. 49 °C); H-NMR: δ 10.23 (1H, s, CHO),
7.45-7.32 (6H, m, Ar-H), 6.96 (1H, d, J = 9.00 Hz, Ar-H), 5.10 (2H, s, PhCH2), 3.89 (3H, s, OCH3);
13C–NMR: δ 190.4 (CH), 152.8 (C), 150.8 (C), 136.3 (C), 129.6 (CH), 129.1 (C), 128.7 (CH), 128.6
(CH), 128.5 (CH), 117.4 (CH), 112.4 (C), 76.5 (CH2), 56.2 (OCH3).
2-Benzyloxy-6-bromo-3-methoxybenzyl alcohol (3b). Standard sodium borohydride reduction of 3a
gave 3b in 94.6% yield as colourless prisms from ethanol, m.p. 84-85°C; Anal.Calc. for C15H15BrO3:
C, 55.8; H, 4.7. Found: C, 55.6; H, 4.9%; 1H-NMR: δ 7.45-7.29 (6H, m, Ar-H), 6.79 (1H, d, J = 9.0
Hz, Ar-H), 5.06 (2H, s, PhCH2), 4.72 (2H, d, J = 5.40 Hz, CH2), 3.87 (3H, s, OCH3), 2.14 (1H, br t, J
13
= 5.40 Hz, OH); C–NMR: δ 152.3 (C), 147.3 (C), 137.0 (C), 134.2 (C), 128.6 (CH), 128.5 (CH),
128.4 (CH), 128.1 (CH), 115.0 (C), 113.3 (CH), 75.8 (CH2), 60.4 (CH2), 56.0 (OCH3).
2-Benzyloxy-6-bromo-3-methoxybenzyl chloride (3c). Treatment of 3b with thionyl chloride gave crude
3c as a white solid in 98.2% yield, m.p. 65-66°C. This was used in the next step without further
1
purification. H-NMR: δ 7.53-7.24 (6H, m, Ar-H), 6.82 (1H, d, J = 9.00 Hz, Ar-H), 5.11 (2H, s,
PhCH2), 4.77 (2H, s, CH2), 3.87 (3H, s, OCH3); 13C–NMR: δ 152.4 (C), 147.6 (C), 137.1 (C), 131.7
(C), 128.5 (CH), 128.4 (CH), 128.3 (CH), 128.2 (CH), 115.4 (C), 114.0 (CH), 75.6 (CH2), 56.1 (OCH3),
41.0 (CH2).
2-Benzyloxy-6-bromo-3-methoxybenzyl cyanide (3d). Treatment of 3c with sodium cyanide in
acetone/water gave 3d as colourless needles in 94.9% yield, m.p. 54-55°C; EI-MS (70 eV) m/z 333
[(M+2)+, 13%], 331 (M+, 14), 91(100); Anal. calc. for C16H14BrNO2: C, 57.9; H, 4.3; N, 4.2. Found: C,
1
57.7; H, 4.5; N, 4.0%; H-NMR: δ 7.48-7.28 (6H, m, Ar-H), 6.83 (1H, d, J = 8.70 Hz, Ar-H), 5.12
13
(2H, s, PhCH2), 3.88 (3H, s, OCH3), 3.75 (2H, s, CH2); C-NMR: δ 152.3 (C), 146.9 (C), 136.7 (C),
128.7 (CH), 128.6 (CH), 128.5 (CH), 128.0 (CH), 125.1 (C), 117.1 (C), 114.7 (C), 113.8 (CH), 75.3
(CH2), 56.1 (OCH3), 19.0 (CH2).

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2-Benzyloxy-6-bromo-3-methoxyphenylacetic acid (3e). Hydrolysis of 3d with potassium hydroxide in
ethanol/water gave 3e as colourless needles in 88.7% yield, m.p. 99-100°C; EI-MS (70 eV) m/z 352
[(M+2)+, 14%], 350 (M+, 14), 270(84), 244(61), 91(100). Anal. calc. for C16H15BrO4: C, 54.7; H, 4.3.
1
Found: C, 54.9; H, 4.1%; H-NMR: δ 7.44-7.26 (6H, m, Ar-H), 6.79 (1H, d, J = 9.00 Hz, Ar-H), 5.02
(2H, s, PhCH2), 3.87 (3H, s, OCH3), 3.84 (2H, s, CH2); 13C–NMR: δ 175.9 (C), 152.1 (C), 147.4 (C),
137.1 (C), 128.8 (C), 128.5 (CH), 128.3 (CH), 128.2 (CH), 127.6 (CH), 116.0 (C), 112.9 (CH), 75.1
(CH2), 56.0 (OCH3), 35.8 (CH2).
2-(2-Benzyloxy-6-bromo-3-methoxyphenyl)-N-(3,4-methylenedioxyphenethyl)acetamide (4).
A
mixture of 2-benzyloxy-6-bromo-3-methoxyphenylacetic acid (3e, 21.5 g) and thionyl chloride (20.3 g)
in benzene (150 mL) was refluxed for 1 h, then the solvent and excess thionyl chloride were removed
in vacuo. The resulting crude 2-benzyloxy-6-bromo-3-methoxyphenylacetyl chloride (3f) was
dissolved in chloroform (200 mL) and added portionwise to a mixture of 2-(3,4-methylene-
dioxyphenyl)ethylamine (2, 11.2 g) [9] in chloroform (100 mL), sodium hydrogen carbonate (25 g)
and ice (200 g). The mixture was stirred at room temperature for 3 h. The chloroform layer was
washed with 10% sodium carbonate (3 x100 mL), water (2 x100 mL), 5% HCl (3 x 100 mL), brine
and then dried. Removal of the solvent under vacuum gave a pale yellow solid which was
recrystallized from ethanol to give amide 4 as a pale yellow solid (21.6 g , 70.8%), m.p. 143-144°C;
UV λmax nm (MeOH) (log ε ) 231sh (4.09), 286 (3.77); IR νmax (KBr): 3290 (NH), 3062, 3031, 3007,
2938, 2888, 2839, 1647 (C=O), 1550, 1504, 1489, 1471, 1441, 1406, 1366, 1344, 1274, 1249, 1129,
1073, 1040, 978, 927, 861, 805, 750, 696 cm-1; EI-MS(70 eV) m/z 499 [(M+2)+, 4%], 497 (M+, 4),
418 (10), 352(7), 270(9), 238(30), 223(14), 164(8), 148(82), 135(19), 91(100). Anal. calc. for
C25H24BrNO5: C, 60.3; H, 4.9; N, 2.8. Found: C, 60.5; H, 4.7; N, 2.7%; 1H-NMR: δ 7.46-7.23 (6H, m,
Ar-H), 6.78 (1H, d, J = 8.70 Hz, Ar-H), 6.61 (1H, d, J = 7.80 Hz, Ar-H), 6.54 (1H, d, J = 1.50 Hz, Ar-
H), 6.47 (1H, dd, J = 7.80, 1.50 Hz, Ar-H), 5.49 (1H, br s, N-H), 4.98 (2H, s, PhCH2), 3.88 (3H, s,
OCH3), 3.68 (2H, s, CH2), 3.34 (2H, apparent q, J = 6.60 Hz, CH2N), 2.60 (2H, t, J = 6.60 Hz, CH2);
13C–NMR: δ 169.3 (C), 152.3 (C) , 147.6 (C), 147.0 (C), 146.0 (C), 137.0 (C), 132.5 (C), 129.7 (C),
128.5 (C), 128.4 (CH), 128.3 (CH), 128.1 (CH), 121.6 (CH), 113.5 (CH), 112.7 (CH), 109.0 (CH),
108.2 (CH), 100.8 (CH2), 75.1 (CH2), 55.9 (OCH3), 40.8 (CH2), 38.4 (CH2), 35.2 (CH2).
1-(2-Benzyloxy-6-bromo-3-methoxybenzyl)-6,7-methylenedioxy-3, 4-dihydroisoquinoline
(5). A
solution of amide 4 (6.03 g) and phosphorus oxychloride (19.4 g) in benzene (50 mL) was refluxed for
2 h. The excess reagent and solvent were removed under vacuum. The resulting brown residue was
shaken with chloroform (80 mL) and dilute ammonium hydroxide (60 mL). The chloroform layer was
washed with water (3 x 60 mL) and then dried. Removal of the solvent under vacuum followed by
recrystallisation from ethanol gave dihydroisoquinoline 5 as pale yellow prisms (5.64 g, 97.1%), m.p.
81-82°C; UV λmax nm (log ε) 227sh (4.52), 280 (3.91), 313 (3.90); IR νmax(KBr): 1636, 1600, 1574,
1266, 1233, 1173, 1129, 1097, 1076, 1038, 1013, 980, 934, 865, 798, 751, 697 cm-1; Anal. calc. for
C25H22BrNO4: C, 62.5; H, 4.6; N, 2.9. Found: C, 62.7; H, 4.4; N, 2.8%; 1H-NMR: δ 7.40-7.26 (6H, m,
Ar-H), 7.25 (1H, s, Ar-H), 6.75 (1H, d, J = 8.70 Hz, Ar-H), 6.63 (1H, s, Ar-H), 5.00 (2H, s, PhCH2),
4.17 (2H, s, CH2), 3.85 (3H, s, OCH3), 3.50 (2H, t, J = 7.20 Hz, CH2), 2.46 (2H, t, J = 7.20 Hz, CH2);
13C–NMR: δ 163.8 (C), 152.2 (C), 148.8 (C), 147.4 (C), 146.3 (C), 137.7 (C), 133.3 (C), 132.6 (C),

Molecules 2009, 14
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128.3 (CH), 128.1 (CH), 127.8 (CH), 127.7 (CH), 123.5 (C), 116.3 (C), 112.0 (CH), 107.8 (CH), 105.6
(CH), 101.2 (CH2), 74.8 (CH2), 55.8 (OCH3), 46.8 (CH2), 37.0 (CH2), 26.3 (CH2).
1-(2-Benzyloxy-6-bromo-3-methoxybenzyl)-6,7-methylenedioxy-1,2,3,4-tetrahydroisoquinoline (6a).
Sodium borohydride (2.0 g) was added portionwise to a stirred solution of dihydroisoquinoline 5 (18.2
g) in ethanol (310 mL) and the mixture was refluxed for 1 h. Chloroform (200 mL) was added and the
mixture was washed with water (4 x 200 mL), brine and then dried. Removal of the solvent under
vacuum gave tetrahydroisoquinoline 6a as a yellow-red viscous oil (14.8 g, 81.0%) which failed to
1
crystallise and was used in the next step without further purification. H-NMR: δ 7.47-7.26 (6H, m,
Ar-H), 6.73 (1H, d, J = 8.70 Hz, Ar-H), 6.66 (1H, s, Ar-H), 6.50 (1H, s, Ar-H), 5.84 (2H, s, OCH2O),
5.05 and 4.92 (each 1H, d, J = 10.80 Hz, PhCH2), 4.23 (1H, dd, J = 10.50, 2.70 Hz, H-1), 3.85 (3H, s,
OCH3), 3.30-2-43 (6H, m, CH2); 13C-NMR: δ 152.1 (C), 147.6 (C), 145.8 (C), 145.6 (C), 137.3 (C),
133.5 (C), 131.9 (C), 128.5 (CH), 128.3 (CH), 128.2 (CH), 128.0 (CH), 127.6 (C), 115.9 (C), 112.0
(CH), 108.5 (CH), 106.9 (CH) 100.5 (CH2), 75.0 (CH2), 55.8 (OCH3), 55.5 (CH), 39.4 (CH2), 37.6
(CH2), 29.9 (CH2).
2-Trifluoroacetyl-1-(2-benzyloxy-6-bromo-3-methoxybenzyl)-6,7-methylenedioxy-1,2,3,4-tetrahydro-
isoquinoline (6b). Trifluoroacetic anhydride (38.5 g) was added dropwise to a stirred mixture of tetra-
hydroisoquinoline 6a (14.8 g) and triethylamine (26.6 g) in chloroform (230 mL) at 0-10°C. Stirring
was continued at room temperature for 3 h. Chloroform (150 mL) was added and the chloroform layer
was washed with 10% sodium hydrogen carbonate (4 x 200 mL), 10% HCl (3 x 250 mL), brine and
then dried. Removal of the solvent gave a red-brown viscous oil which crystallized from ethanol to
give trifluoroacetamide 6b as a pale yellow solid (7.0 g, 39.4%); m.p. 122-123°C; UV λmax nm
(MeOH) (log ε ) 231sh (4.17), 289 (3.80); IR νmax (KBr): 1681, 1571, 1504, 1300, 1284, 1269, 1235,
1190, 1180, 1158, 1082, 1036, 984, 976, 961, 941, 912, 855, 795, 759, 699 cm-1; 1H-NMR (d6-DMSO):
δ 7.47-7.31 (5H, m, Ar-H), 7.23 (1H, d, J = 8.70 Hz, Ar-H), 6.82 (1H, d, J = 8.70 Hz, Ar-H), 6.54 (1H,
s, Ar-H), 6.37 (1H, s, Ar-H), 5.91 (2H, s, OCH2O), 5.68 (1H, dd, J = 10.70, 3.20 Hz, H-6a), 5.16 and
4.95 (each 1H, d, J = 10.80 Hz, PhCH2), 3.88 (3H, s, OCH3), 3.85-3.65 (1H, m, CH), 3.30-2.54 (5H, m,
CH2); 13C–NMR: (d6-DMSO): δ 155.1 (C), 151.6 (C), 147.8 (C), 146.6 (C), 146.2 (C), 137.2 (C),
130.7 (C), 128.4 (CH), 128.3 (CH), 128.2 (CH), 128.1 (CH), 127.1 (C), 125.8 (C), 118.2 (C), 115.5
(C), 113.0 (CH), 108.0 (CH), 106.6 (CH), 100.8 (CH2), 74.7 (CH2), 55.9 (OCH3), 53.4 (CH), 39.8
(CH2), 36.6 (CH2), 29.0 (CH2).
8-Benzyloxy-9-methoxy-1,2-methylenedioxy-6-trifluoroacetylnoraporphine (1b). A solution of 2,2′-
azobis(isobutyronitrile) (1.59 g) and tributyltin hydride (6.18 g) in toluene (70 mL) was added in 6
equal portions over 2.5 h to a refluxing solution of trifluoroacetamide 6b (6.23 g) in toluene (110
mL). The resulting mixture was then refluxed for 24 h. The solvent was removed under vacuum and
the resulting yellow residue was dissolved in acetonitrile (500 mL) and washed with hexane (5 x 200
mL) and then dried. Removal of the solvent under vacuum gave a yellow solid which was triturated
from ethanol to give noraporphine 1b as colourless needles (2.15 g, 40.1%); m.p. 218 -219°C; UV
λ
max nm (log ε ) 282 (4.33), 323sh (3.71); IR νmax (KBr): 1681 (C=O), 1601, 1573, 1273, 1245, 1236,
1215, 1202, 1187, 1173, 1153, 1136, 1080, 1061, 1037, 944, 919, 847, 816, 735, 694, 651 cm-1; EI-

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MS (70 eV) m/z 497(M+, 91%), 406(91), 374(45), 309(24), 91(100). Anal. calc. for C27H22F3NO5: C,
1
65.2; H, 4.5; N, 2.8. Found: C, 65.0; H, 4.7; N, 3.0%; H-NMR: δ7.86 (1H, d, J = 8.70 Hz, H-11),
7.60-7.25 (5H, m, Ar-H), 6.91 (1H, d, J = 8.70 Hz, H-10), 6.54 (1H, s, H-3), 6.02 (2H, AB q, J = 1.20
Hz, OCH2O), 5.10 and 4.97 (each 1H, d, J = 10.80 Hz, PhCH2), 4.94-4.83 (1H, m, H-6a), 4.18 (1H, br
13
d, J = 13.20 Hz, Ha-5), 3.91 (3H, s, OCH3), 3.63-2.35 (5H, m, CH2); C–NMR: δ 155.8 (C), 152.6
(C), 147.1 (C), 145.2 (C), 142.8 (C), 137.6 (C), 129.6 (C), 128.6 (CH), 128.4 (CH), 128.0 (CH), 126.4
(C), 123.8 (C), 123.6 (CH), 118.3 (C), 117.5 (C), 114.5 (C), 110.6 (CH), 106.7 (CH), 101.0 (CH2),
75.1 (CH2), 55.8 (OCH3), 52.3 (CH), 41.4 (CH2), 30.4 (CH2), 26.8 (CH2).
8-Hydroxy-9-methoxy-1, 2-methylenedioxy-6-trifluoroacetylnoraporphine (1c).
A solution of
noraporphine 1b (2.0 g) in methanol (100 mL) and chloroform (250 mL) was hydrogenolysed in the
presence of 10% Pd/C (2.8 g) at 45-50 psi for 4 h. The catalyst was filtered off and the solvent
removed under vacuum. The resulting white residue was shaken with chloroform (150 mL) and 10%
ammonium hydroxide (100 mL). Removal of the solvent under vacuum followed by recrystallisation
form ethanol gave noraporphine 1c as pale brown needles (0.65 g , 40.0%); m.p. 287-289 °C; UV λmax
nm (log ε ) 284 (4.24), 298 (4.12), 321sh (3.81); IR νmax (KBr): 3459 (OH), 1674 (C=O), 1613, 1583,
1275, 1240, 1196, 1186, 1175, 1161, 1145, 1078, 1061, 1023, 940, 916, 897, 845, 810, 764, 757
cm-1; EI-MS(70 eV) m/z 407(M+,74%), 376(13), 346(30), 327(20), 305(64), 281(100), 272(39),
256(39), 206(28), 167(30), 91(100); Anal. calc. for C20H16F3NO5: C, 59.0; H, 4.0; N, 3.4. Found: C,
1
59.2; H, 3.8; N, 3.2%; H-NMR: δ 7.69 (1H, d, J = 8.70 Hz, H-11), 6.87 (1H, d, J = 8.70 Hz, H-10),
6.59 (1H, s, H-3), 6.06 (2H, AB q, J = 1.20 Hz, OCH2O), 5.13 (1H, dd, J = 13.50, 4.50 Hz, H-6a),
4.30-4.18 (1H, m, Ha-5), 3.96 (3H, s, OCH3), 3.66-2.48 (5H, m, CH2); 13C-NMR (d6-DMSO): δ 154.6
(C), 146.9 (C), 146.0 (C), 142.6 (C), 141.4 (C), 127.8 (C), 126.8 (C), 124.1 (C), 122.4 (C), 118.1
(CH), 116.1(C), 109.0 (CH), 106.8 (CH), 100.1 (CH2), 55.6 (OCH3), 53.0 (CH), 43.0 (CH2), 29.1
(CH2), 29.0 (CH2).
(±)-Norannuradhapurine (1a). Noraporphine 1c (0.77 g) was dissolved in methanol (140 mL) with
heating. Potassium carbonate (1.46 g) in water (4.5 mL) was added and then the mixture was refluxed
for 2.5 h. The solvent was removed under vacuum. The resulting residue was shaken with water (60
mL) and 10% sodium carbonate (40 mL), and extracted with chloroform (4 x 40 mL) and then dried.
Removal of the solvent under vacuum followed by recrystallisation form ethanol gave
(±)norannuradhapurine (1a) as purple needles (0.46 g, 78.2%) ; m.p. 207-209°C; UV λmax nm (log
ε ) 217 (4.43), 283 (4.24), 298sh (4.09), 320sh (3.76); IR νmax (KBr): 3276 (OH), 2583, 1608, 1578,
1288, 1258, 1235, 1167, 1146, 1127, 1083, 1059, 1023, 991, 947, 909, 841, 792 cm-1; EI-MS(70 eV)
m/z 311(M+, 54%), 310 (100), 278(17), 91(14); Anal. calc. for C18H17NO4: C, 69.4; H, 5.5; N, 4.5.
Found: C, 69.6; H, 5.3; N, 4.6%; 1H-NMR: δ 7.64 (1H,d, J = 8.40 Hz, H-11), 6.82 (1H, d, J = 8.40 Hz,
H-10), 6.54 (1H, s, H-3), 6.00 (1H, AB q, J = 1.50 Hz, 2H, OCH2O), 3.92 (3H, s, OCH3), 3.88 (1H, dd,
J = 13.80, 5.10 Hz, H-6a), 3.45-2.34 (6H, m, CH2); 13C-NMR (CDCl3): δ 146.5 (C), 145.9 (C), 142.2
(C), 142.0 (C), 127.8 (C), 126.8 (C), 125.0 (C), 121.5 (C), 118.8 (CH), 116.4 (C), 108.4 (CH), 107.3
13
(CH), 100.5 (CH2), 56.0 (OCH3), 53.3 (CH), 43.5 (CH2), 29.6 (CH2), 29.2 (CH2); C NMR (d6-
DMSO) δ/ppm 147.0 (C), 145.9 (C), 142.7 (C), 141.4 (C), 128.0 (C), 126.9 (C), 124.2 (C), 122.6 (C),

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118.1 (CH), 116.1 (C), 109.4 (CH) , 106.8 (CH), 100.1 (CH2), 55.8 (OCH3), 53.0 (CH), 43.0 (CH2),
29.2 (CH2), 29.1 (CH2).
Minimum inhibitory concentration (MIC)
MIC of (±)-norannuradhapurine was determined by NCCLS microbroth dilution methods [12]. (±)-
norannuradhapurine was weighed and dissolved in DMSO to make a solution of concentration 2.56
mg/mL. From this stock solution two-flow serial dilution has been carried out to give a series of
solutions from 256 μg/mL to 0.50 μg/mL with culture medium in 96-well microplates (100 μl of total
volume). Three different microorganisms were selected viz. Staphytolcoccus aureus ATCC25932,
Escherichia coli ATCC10536 and Candida albicans ATCC90028. They were subcultured on nutrient
broth supplemented with 10% glucose (NBG) (for bacteria) or Sabouraud glucose broth (for yeast) and
incubated at 37 °C for 24 h. A final concentration of 1 x 105 cfu/mL of test bacteria or yeast was added
to each dilution. The plates were incubated at 37 °C for 48 h. MIC was defined as the lowest
concentration of test agent that inhibited bacterial or yeast growth, as indicated by the absence of
turbidity. Test agent-free broth containing 5% DMSO was incubated as growth control.
Anti-inflammatory activity
Murine macrophage RAW 264.7 cell line obtained from American Type Culture Collection
(ATCC, Maryland, USA), was maintained in DMEM supplemented with 10% heat inactivated FBS,
penicillin G (100 IU/mL), streptomycin (100 mg/mL), and L-glutamine (2 mM) and incubated at 37 ºC
in a humidified atmosphere containing 5% CO2. Cells (1 x 106 /mL) were pre-incubated 2 h with (±)-
norannuradhapurine (1, 2.5 and 5 μg/mL) and further cultured 24 h with LPS (1 μg/mL) in 24-well
plates. Supernatants were removed at the allotted times and NO, PGE2, TNF-α, IL-1β and IL-6 levels
were quantified by immunoassay kits according to the manufacture’s protocols (Assay Designs’
Correlate-EIATM, Stressgen, USA), respectively. Western blot Cellular proteins were extracted from
control and (±)-norannuradhapurine-treated RAW 264.7 cells. The washed cell pellets were
resuspended in lysis buffer buffer (50 mM HEPES pH 7.0, 250 mM NaCl, 5 mM EDTA, 0.1%
Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 5 mM NaF, 0.5 mM Na
orthovanadate) containing 5 μg/mL each of leupeptin and aprotinin and incubated for 15 min at 4 ºC.
Cell debris was removed by microcentrifugation, followed by quick freezing of the supernatants.
Protein concentration was determined by BioRad protein assay reagent according to the manufactures
instruction, 40-50 μg of cellular proteins from treated and untreated cell extracts were electroblotted
onto nitrocellulose membrane following separation on a 10% SDS-polyacrylamide gel electrophoresis.
The immunoblot was incubated overnight with blocking solution (5% skim milk) at 4 ºC, followed by
incubation for 4 h with a 1:500 dilution of monoclonal anti-iNOS and COX-2 antibody (Santacruz,
CA, USA). Blots were washed 2 times with PBS and incubated with a 1:1000 dilution of horseradish
peroxidase-conjugated goat anti-mouse IgG secondary antibody (Santacruz, CA, USA) for 1 h at room
temperature. Blots were again washed three times in Tween 20/Tris-buffered saline (TTBS) and then
developed by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL, USA).

Molecules 2009, 14
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Cytotoxicity assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity
assay was performed according to the method previously described [13]. MTT solution was added at a
concentration of 50 μg/mL into each well, which also contain 1, 2.5 and 5 μg/mL of (+)-3-
methoxynordomesticine. After 4 h of incubation at 37 ºC, the medium was discarded and the formazan
blue, which formed in the cells, was dissolved in 50 μl DMSO. The optical density at 540 nm was
determined with a microplate reader. The optical density of formazan formed in control (untreated)
cells was taken as 100% of viability.
Acknowledgements
The authors are grateful to Mrs. Panit Vedkanchana and Mr. Witoon Ngow of the Silpakorn
University Scientific and Technological Research Equipment Centre for kindly measuring the low
resolution mass spectra.
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