Total syntheses of (±)-gusanlung A, (±)-gusanlung D and 8-oxyberberrubine and the uncertainty concerning the structures of (-)-gusanlung A, (-)-gusanlung D and 8-oxyberberrubine

Article abstract of DOI:10.3390/molecules14020726

(±)-Gusanlung A, 8-oxyberberrubine and (±)-gusanlung D have been synthesized by radical cyclisation of the corresponding 2-aroyl-1-methylenetetrahydroisoquinolines. The ¹H and ¹³C spectra of (-)-gusanlung D were found to be different

Full text of DOI:10.3390/molecules14020726

Molecules 2009, 14, 726-737; doi:10.3390/molecules14020726
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Total Syntheses of (±)-Gusanlung A, (±)-Gusanlung D and 8-
Oxyberberrubine and the Uncertainty Concerning the
Structures of (-)-Gusanlung A, (-)-Gusanlung D and 8-
Oxyberberrubine
Surachai Nimgirawath 1,*, Phansuang Udomputtimekakul 1, Thitima Apornpisarn 1, Asawin
Wanbanjob 1 and Thongchai Taechowisan 2
1
Department of Chemistry, Faculty of Science, Silpakorn University, Nakorn Pathom 73000,
Thailand; E-mails: Phansuang@yahoo.com (P.U.), winsusc@gmail.com (T.A.),
thitima_noon@hotmail.com (T.A.)
2
Department of Microbiology, Faculty of Science, Silpakorn University, Nakorn Pathom 73000,
Thailand; E-mail: tthongch@su.ac.th (T.T.)
* Author to whom correspondence should be addressed; E-mail: surachai@su.ac.th
Received: 12 January 2009; in revised form: 9 February 2009 / Accepted: 12 February 2009 /
Published: 12 February 2009
Abstract: (±)-Gusanlung A, 8-oxyberberrubine and (±)-gusanlung D have been
synthesized by radical cyclisation of the corresponding 2-aroyl-1-methylenetetra-
1
13
hydroisoquinolines. The H and C spectra of (-)-gusanlung D were found to be different
from those of synthetic (±)-gusanlung D. Careful analyses of the 13C spectra of (–)-
gusanlung A and natural 8-oxyberberrubine also cast doubt on the correctness of the
structures previously assigned to these two compounds. (±)-Gusanlung A and (±)-
gusanlung D were inactive against Staphylococcus aureus ATCC25932, Escherichia coli
ATCC10536 and Candida albicans ATCC90028.
Keywords: Alkaloid; Protoberberine; Isoquinoline; Synthesis; Antimicrobial activity.

Molecules 2009, 14
727
Introduction
(–)-Gusanlung D, isolated from Acangelisia gusanlung H. S. Lo (Menispermaceae), is the first
natural 8-oxotetrahydroprotoberberine alkaloid with an unoxygenated ring D [1]. Based on spectral
data analysis, structure 1 was proposed for (–)-gusanlung D. Prior to the isolation of (–)-gusanlung D,
Kessar et al. synthesized in 1992 a compound which is essentially (±)-gusanlung D [2]. However, a
1
close comparison of the H-NMR data of (±)-gusanlung D with those reported for (–)-gusanlung D
revealed significant differences. In 2003 Reimann, Grasberger and Polborn reported another synthesis
of (±)-gusanlung D [3]; in this case the 13C-NMR spectral data were found to show significant
differences to those reported for (–)-gusanlung D. Subsequently, an unsymmetric synthesis of (–)-
gusanlung D was achieved by Chrzanowska, Dreas and Razwadowska in 2004 [4]. Comparison of the
1H- and 13C-NMR data of synthetic (–)-gusanlung D with those of natural (–)-gusanlung D also
showed significant differences. Finally, Chang and Chang reported a total synthesis of (±)-gusanlung
D [5], whose spectral data were said to agree with those in references [1-4]. This last conclusion added
further confusion to the matter since, if the spectral data of (±)-gusanlung D [5] are in good agreement
with those reported for (±)-gusanlung D [2-3] and synthetic (–)-gusanlung D [4], they cannot also be
consistent with those reported for natural (–)-gusanlung D [1]. In view of these discrepancies in the
1H- and 13C-NMR data of natural (–)-gusanlung D [1] and the synthetic alkaloids, it was therefore
highly desirable to perform another independent synthesis of (±)-gusanlung D to shed further light on
the possible structure of (–)-gusanlung D.
Figure 1. Structures of (–)-gusanlung D (1), (–)-gusanlung A (2) and 8-oxyberberrubine (3).
4
1
5
4 a
1 4 a
3
2
O
O
O
O
6
8
N
O
N
O
8 a
1 4
H
O H
R
R
1 3
1
2
9
1 2 a
1 2
O C H
1 0
3
1 1
(1) R1 = R2 = H
(2) R1 = OH, R2 = OCH3
(3)
Furthermore, two new related alkaloids: (–)-gusalung A [6] and 8-oxyberberrubine [1], for which
structures 2 and 3 were proposed based on spectral analysis, were isolated from Acangelisia gusanlung
H. S. Lo. In view of the uncertainty regarding the correct structure of (–)-gusanlung D (1), it was
therefore highly desirable to also confirm the correctness of the structures proposed for (–)-gusanlung
A (2) and 8-oxyberberrubine (3) by total syntheses.

Molecules 2009, 14
728
Results and Discussion
Syntheses of (±)-gusanlung A (2) and 8-oxyberberrubine (3)
The synthesis of (±)-gusanlung A (2) was based on the radical-initiated cyclization of 2-(2′-
benzyloxy-6′-bromo-3′-methoxybenzoyl)-1-methylene-6,7-methylenedioxy-1,2,3,4-tetrahydroisoqui-
noline (6a), as outlined in Scheme 1, with subsequent catalytic hydrogenolysis of the benzyl protecting
group.
Scheme 1. Synthetic routes to (±)-gusanlung A (1), (±)-gusanlung D (2), and 8-oxyberberrubine (3).
R
1
O
O
R
R
R
C
2
4
3
N
O
+
O
N
5 + 4 c
6 a
6 b
O
5 + 4 d
5
C H
3
R
R
3
1
4 a : R = O B n , R = O C H
R = B r , R = C H O
4 b : R 1 = O B n , R 2 = O C H 3 , R 3 = B r , R 4 = C O O H
A
B
R
2
1
2
3 ,
3 ,
3
4
4 c : R = O B n , R = O C H
R
= B r , R = C O C l
4 d : R 1 = R = H ,2 R = I , R =3 C O C l
4
6 a : R = O B n , R = O C H
6 b : R 1 = R = H ,2 R = I
R
= B r
3 ,
3
1
2
3
4
1
2
3
O
O
O
O
O
O
N
O
N
O
N
O
E
F
( 7 a + 8 a
( 1 + 8 b
2 )
1 )
R
R
R
R
1
2
R
R
1
2
D
1
2
8 a : R = O B n ,
1
7 a : R = O B n , R = O C H
1
1
1
2
3
R
= O C H
3
8 b : R2 = R = H
2 : R = O H , R = O C H
[ (1 ) - g u s a n lu n g A ]
2
3
: R = R = H
2
1
2
+
1
+
1 : R = R = H [ ( ) - g u s a n lu n g D ]
2
G ( 7 a + 8 a
8 a )
O
O
O
O
N
O
N
O
H
O B n
O C H
O H
O C H
3
3
8 a
3 : [ 8 - o x y b e r b e r r u b in e ]
Reagents and Conditions A) NaClO2, sulphamic acid/ tert-butanol-H2O; B) SOCl2/ benzene; C)
Et3N/ dry benzene; D) Bu3SnH, AIBN/ dry benzene; E) H2, Pd/C/ ethanol; F) hydrazine, Pd/C/
ethyl acetate-ethanol; G) I2/ dioxane; H) conc. HCl/ ethanol.
Thus, oxidation of 2-benzyloxy-6-bromo-3-methoxybenzaldehyde (4a) [7] with sodium chlorite
gave 2-benzyloxy-6-bromo-3-methoxybenzoic acid (4b), whose acid chloride (4c) was then reacted

Molecules 2009, 14
729
with 6,7-methylenedioxy-1-methyl-3,4-dihydroisoquinoline (5) [8] in the presence of triethylamine to
give thee moderately stable compound 6a. Treatment of 6a with tributyltin hydride in the presence of a
catalytic amount of 2,2′-azobis(isobutyronitrile) gave a 31.3% yield of a mixture of (±)-9-
1
benzylgusanlung A (7a) and 9-benzyl-8-oxyberberrubine (8a) in a ratio of 78:22 according to H-
NMR analysis. Catalytic hydrogenolysis of the mixture of 7a and 8a to remove the benzyl protecting
group also resulted in the concurrent hydrogenation of the C-C double bond to give pure (±)-gusanlung
A (2). On the other hand, oxidation of the mixture of 7a and 8a with iodine gave 9-benzyl-8-
oxyberberrubine (8a), whose benzyl protecting group was removed by acid treatment to give 8-oxy-
berberrubine (3).
The 1H-NMR data of synthetic (±)-gusanlung A (2) were in reasonably good agreement with those
reported for natural (–)-gusanlung A (2). However, a number of carbons in the 13C-NMR spectrum of
natural (–)-gusanlung A (2) were found to have quite different chemical shifts from the corresponding
1
carbons in the spectrum of (±)-gusanlung A (2). We therefore carried out H-1H-COSY, HMQC and
HMBC experiments to allow complete assignments of chemical shifts of (±)-gusanlung A (2). Details
of the HMBC correlations are shown in Figure 2 and Table 4. The 1H-NMR spectral data of natural 8-
oxyberberrubine (3) were found to be in good agreement with those of synthetic 8-oxyberberrubine
(3). However, from HMBC correlation experiment, it was possible to establish that the chemical shifts
13
of H-1 and H-13 previously assigned should be interchanged. On the other hand, the C spectrum of
natural 8-oxyberberrubine (3) had a number of features which were quite different from those of
synthetic 8-oxyberberrubine (3). These differences were highlighted and the HMBC correlations were
shown in Figure 2 and Table 5. In summary, it can be concluded that while the 1H-NMR analysis lent
good support to the structures proposed for (–)-gusanlung A (2) and 8-oxyberberrubine (3), in view of
the discrepancies in a number of carbon chemical shifts in the 13C-NMR spectra of (-)-gusanlung A (2)
versus those of (±)-gusanlung A (2) on the one hand, and natural 8-oxyberberrubine (3) versus
synthetic 8-oxyberberrubine (3) on the other, no definite conclusions can be drawn at this time
concerning the correctness of the structures previously assigned to (–)-gusanlung A (2) and 8-
oxyberberrubine (3).
Figure 2. HMBC correlations of (±)-gusanlung A (1) and 8-oxyberberrubine (3).
O
O
5
O
O
4
1
5
4
3
4 a
1 4 a
6
3
4 a
1 4 a
6
2
2
N
O
1
N
O
H
O
1 4
1 3
8
1 4
1 3
1 2 a
8
8 a
9
8 a
9
O H
1 2 a
1 2
1 2
1 0
1 0
O C H
O C H
3
3
1 1
1 1
1
3

Molecules 2009, 14
730
Synthesis of (±)-gusanlung D
The synthesis of (±)-gusanlung D (1) was uneventful. Thus, 2-iodobenzoyl chloride (4d) was
reacted with 5 [8] in the presence of triethylamine to give the highly unstable 2-(2′-iodobenzoyl)-1-
methylene-6,7-methylenedioxy-1,2,3,4-tetrahydroisoquinoline (6b). Treatment of 6b with tributyltin
hydride in presence of a catalytic amount of 2,2′-azobis(isobutyronitrile) gave a 39.0% yield of a
1
mixture of 1 and 8b in a ratio of 87:23 from H-NMR analysis. Treatment of the mixture with
1
13
hydrazine and palladium/charcoal gave (±)-gusanlung D (1), whose H- and C-NMR data were in
good agreement with those of (±)-gusanlung D (1) and (–)-gusanlung D obtained from previous
syntheses [2,3,4] but differed significantly from those of natural (–)-gusanlung D [1]. The structure
previously assigned to (–)-gusanlung D [1] therefore remains uncertain.
Table 1. Comparison of 1H-NMR spectral data between natural (-)-gusanlung D [1],
synthetic (-)-gusanlung D [4] and synthetic (±)-gusanlung D [2] and [this work].
(–)-gusanlung D
CDCl3 [1]
(–)-gusanlung D
CDCl3 [4]
(±)-gusanlung D
CDCl3 [2]
(±)-gusanlung D
CDCl3 [this work]
m.p. 175-176 °C
1H
(position)
m.p. 250-251 °C
1H
m.p. 195-197 °C
1H
m.p. 175-177 °C
1H
1
4
7.35 (s)
6.71 (s)
6.76 (d)
6.72 (s)
6.80 (s)
6.67 (s)
6.76 (d)
6.67 (s)
5α
2.70-3.40 (m)
2.70-3.40 (m)
2.70-3.40 (m)
4.8 (m)
2.7-2.8 (m)
2.83-3.35 (m)
2.83-3.35 (m)
2.83-3.35 (m)
4.7-5.1 (m)
8.1-8.37 (m)
7.25-7.65 (m)
7.25-7.65 (m)
7.25-7.65 (m)
2.83-3.35 (m)
2.83-3.35 (m)
4.7-5.1 (m)
5.93 (s)
2.70-2.82 (m)
2.87-3.07 (m)
2.87-3.07 (m)
4.88-4.99 (m)
8.13 (dd, 7.6, 1.4)
7.39 (br t, 7.4)
7.46 (dt, 7.4, 1.5)
7.22-7.29 (m)
2.87-3.07 (m)
3.18 (dd, 15.7, 3.7)
4.84 (dd, 13.3, 3.7)
5.96 (s)
5β
2.82-3.02 (m)
2.82-3.02 (m)
4.93-4.99 (m)
8.13 (d, 7.4)
7.34-7.40 (m)
7.41-7.49 (m)
7.24 (d, 7.4)
2.82-3.02 (m)
3.18 (dd, 15.3, 3.7)
4.83 (dd, 13.3, 3.7)
5.96 (s)
6α
6β
9
8.07 (d, 8.0)
7.29-7.41 (m)
7.29-7.41 (m)
7.29-7.41 (m)
2.70-3.40 (m)
2.70-3.40 (m)
3.95 (m)
10
11
12
13α
13β
14
OCH2O
6.20, 6.06 (s)
Table 2. Comparison of 13C-NMR spectral data between natural (-)-gusanlung D [1],
synthetic (-)-gusanlung D [4] and synthetic (±)-gusanlung D [3] and [this work].
(–)-gusanlung D
CDCl3 [1]
m.p. 250-251 °C
13C
(–)-gusanlung D
CDCl3 [4]
m.p. 195-197 °C
13C
(±)-gusanlung D
CDCl3 [3]
m.p. 175-177 °C
13C
(±)-gusanlung D
CDCl3 [this work]
m.p. 175-176 °C
13C
(position)
1
2
3
4
107.3
105.8
146.5b
146.7b
105.97
105.9
146.6c
146.8c
135.0
146.57
147.0
146.77
107.5
108.6
108.81
108.7

Molecules 2009, 14
731
Table 2. Cont.
(–)-gusanlung D
(–)-gusanlung D
(±)-gusanlung D
CDCl3 [3]
m.p. 175-177 °C
128.85
(±)-gusanlung D
CDCl3 [this work]
m.p. 175-176 °C
128.9
(position)
CDCl3 [1]
m.p. 250-251 °C
126.5
CDCl3 [4]
m.p. 195-197 °C
128.8
4a
5
29.7
29.6
29.61
29.7
6
42.0
38.7
38.49
38.8
8
162.0
164.5
158.67
164.6
8a
117.3
137.2
137.24
137.3
9
128.7a
127.9a
127.1a
126.8a
124.6
128.6
128.60
128.6
127.4*
131.9*
10
127.3
127.37
11
131.8
132.33
12
126.8
126.87
126.9
12a
13
129.0
131.81
129.1
33.5
38.1
37.78
38.1
14
49.4
55.2
55.18
55.3
14a
OCH2O
126.5
128.5
128.55
128.6
100.9
101.1
101.00
101.1
a, b, c, * assignments may be interchangeable.
Table 3. Comparison of 1H-NMR spectral data between natural (-)-gusanlung A [1] and
synthetic (±)-gusanlung A [this work].
(-)-gusanlung A
(DMSO-d6) [6]
m.p. 260-262 °C
1H
(±)-gusanlung A
(DMSO-d6) [this work]
m.p. 188-189 °C
1H
(±)-gusanlung A
(CDCl3) [this work]
m.p. 188-189 °C
1H
(position)
1
4
6.96 (s)
7.00 (s)
6.71 (s)
6.80 (s)
6.79 (s)
6.66 (s)
5
2.73-2.81 (m)
2.73-2.81 (m)
4.71 (m)
2.75-2.89 (m)
2.89-3.01 (m)
4.69-4.59 (m)
7.09 (d, 8.1)
6.71 (d, 8.1)
3.36 (dd, 15.2, 3.6)
2.66-2.75 (m)
4.84 (dd, 13.3, 3.4)
3.78 (s)
2.72-2.84 (m)
2.94-3.40 (m)
4.80-4.87 (m)
6.94 (d, 8.1)
6.63 (d, 8.1)
3.14 (dd, 15.2, 3.8)
2.80-2.94 (m)
4.80 (dd, 13.6, 3.5)
3.90 (s)
6α
6β
11
6.99 (d, 8.1)
6.86 (d, 8.1)
3.13 (dd, 15.3, 3.1)
2.62 (dd, 15.3, 13.3)
4.68 (dd, 13.3, 3.1)
3.76 (s)
12
13α
13β
14
C10-OCH3
OCH2O
OH
5.98, 5.99 (s)
-
5.98, 6.00 (s)
12.88 (s)
5.96 (s)
12.83 (s)

Molecules 2009, 14
Table 4. Comparison of 13C-NMR spectral data between natural (-)-gusanlung A [6] and
732
synthetic (-)-gusanlung A [this work] and HMBC correlations of (±)-gusanlung A [this
work].
(-)-gusanlung A
(±)-gusanlung A
(DMSO-d6) [this
work]
m.p. 188-189 °C
13C
(±)-gusanlung A
(CDCl3) [this
work]
m.p. 188-189 °C
13C
(±)-gusanlung A
(DMSO-d6) [6]
(DMSO-d6) [this work]
(position)
HMBC
m.p. 260-262 °C
13C
2J
3J
1
106.1
145.9a
147.7a
107.8
129.1b
29.0
106.6
105.8
C-2
C-3, 4a, 14
2
146.7c
146.5c
108.7
146.8*
146.7*
108.6
-
-
3
-
-
4
C-3
C-2, 5, 14a
4a
128.3
128.1
-
-
5
28.9
29.4
C-4a, 6
C-4, 14a
6
37.8
38.5
38.4
C-5
C-4a, 8, 14
8
8a
161.4
122.3b
149.7a
145.7a
118.9
122.1
128.2b
37.7
168.4
168.6
-
-
111.4
111.4
-
-
9
151.4
151.8
-
-
10
147.2
147.5
-
-
11
116.7
115.4
C-10
C-9, 12a
12
116.9
116.1
C-11
C-8a, 10, 13
12a
13
129.6
128.7
-
-
35.9
37.1
C-12a, 14
C-8a, 12, 14a
14
54.4
55.4
55.7
C-13, 14a
-
-
14a
C10-OCH3
OCH2O
OH
129.3b
60.5
129.1
128.4
-
-
56.3
56.3
C-10
C-2, 3
C-8a, 10
100.5
101.3
101.2
-
C-9
a, b, c, * assignments may be interchangeable.
Table 5. Comparison of 1H- and 13C-NMR spectral data between natural 8-oxyberberubine
(3) [1], synthetic 8-oxyberberubine (3) [this work] and HMBC correlations of 8-
oxyberberrubine [this work].
natural 8-oxy-
berberrubine (3)
CDCl3 [1]
synthetic 8-oxy-
natural 8-oxy-
synthetic 8-oxy-
synthetic
berberrubine (3) berberrubine (3) berberrubine (3)
8-oxyberberrubine (3)
(CDCl3) [this work]
CDCl3 [this
work]
CDCl3 [1]
CDCl3 [this
work]
(position)
HMBC
m.p. 240-241 °C
1H
m.p. 238-239 °C m.p. 240-241 °C m.p. 238-239 °C
1H
13C
13C
2J
C-2
-
3J
1
2
6.83 (s)
7.21 (s)
104.0
141.6
146.4
107.1
109.6
28.4
104.8
147.5*
148.6*
108.0
129.5
28.4
C-3, 4a, 14
-
3
-
-
4
6.72 (s)
6.71 (s)
C-3
-
C-2, 5, 14a
-
4a
5
2.91 (t, 7.2)
4.27 (t, 7.2)
2.92 (t, 6.1)
4.27 (t, 6.1)
C-4a,
6
C-4, 14a
6
39.1
39.1
C-5
C-4a, 8, 14

Molecules 2009, 14
733
Table 5. Cont.
natural 8-oxy-
synthetic 8-oxy-
natural 8-oxy-
synthetic 8-oxy-
synthetic
berberrubine (3)
CDCl3 [1]
berberrubine (3) berberrubine (3) berberrubine (3)
8-oxyberberrubine (3)
(CDCl3) [this work]
CDCl3 [this
work]
CDCl3 [1]
CDCl3 [this
work]
(position)
HMBC
m.p. 240-241 °C
m.p. 238-239 °C m.p. 240-241 °C m.p. 238-239 °C
8
8a
9
164.0
129.9
149.0
147.5
114.9
120.0
128.9
103.6
165.4
111.0
150.3
144.9
119.1
115.3
130.5
103.6
-
-
-
-
-
-
-
10
11
12
12a
13
-
7.30 (AB q, 8.0)
7.00 (AB q, 8.0)
7.28 (d, 8.5)
6.99 (d, 8.5)
C-10
C-9, 12a
C-11 C-8a, 10, 13
-
-
7.21 (s)
6.83 (s)
C-14
C-8a, 12,
14a
14
14a
133.6
122.1
56.7
100.6
-
134.6
123.5
56.7
101.5
-
-
-
-
-
-
-
-
C10-OCH3
OCH2O
OH
3.96 (s)
6.02 (s)
-
3.97 (s)
6.02 (s)
13.14
C-10
C-2, 3
-
* assignments may be interchangeable.
Antimicrobial activity
(±)-Gusanlung D (1) and (±)-gusanlung A (2) at the concentration value 256 μg/mL were inactive
against Staphylococcus aureus ATCC25932, Escherichia coli ATCC10536 and Candida albicans
ATCC90028.
Conclusions
Based on spectral analysis, there were significant discrepancies between the spectral data of natural
(-)-gusanlung D and synthetic (±)-gusanlung D. Hence, the structure previously proposed for (-)-
1
gusanlung D remains doubtful. While the H spectral data of natural (-)-gusanlung A and 8-
oxyberberrubine were in reasonably good agreement with those of synthetic (±)-gusanlung A and 8-
oxyberberrubine, the 13C spectral data of natural (-)-gusanlung A and 8-oxyberberrubine were not
entirely in good agreement with those of synthetic (±)-gusanlung A and 8-oxyberberrubine. The
structures previously proposed for natural (-)-gusanlung A and 8-oxyberberrubine must therefore be
treated with caution.
Experimental
General
Melting points were determined on a SMP 2 Stuart Scientific melting point apparatus and are
uncorrected. Infrared spectra were recorded on CH2Cl2-films with a Perkin Elmer Spectrum GX FT-IR

Molecules 2009, 14
734
spectrophotometer. Ultraviolet spectra were recorded on methanol solutions with a Perkin Elmer
1
13
Lambda 35 UV-VIS spectrophotometer. H- and C-NMR spectra were recorded on (D) chloroform
1
solutions at 300 MHz for H and 75 MHz for 13C with a Bruker AVANCE 300 spectrometer.
Tetramethylsilane was used as the internal standard. MS spectra were recorded on a POLARIS Q mass
spectrometer.
2-Benzyloxy-6-bromo-3-methoxybenzoic acid (4b). A solution of sodium chlorite (0.36 g, 3.6 mmol) in
H2O (5 mL) was added to a solution of 2-benzyloxy-6-bromo-3-methoxybenzaldehyde (4a) [7] (1.0 g,
3.1 mmol) and sulfamic acid (0.5 g) in tert-butanol (10 mL) and H2O (3 mL). The solution was stirred
for 1 h. The mixture was shaken with ethyl acetate (20 mL) and the ethyl acetate layer was extracted
with 5% sodium carbonate (3 × 20 mL). The aqueous layer was then acidified with concentrated
hydrochloric acid and extracted with chloroform (3 × 20 mL). The chloroform layer was dried over
anhydrous sodium sulfate. Removal of the solvent under vacuum gave a solid which was recrystallized
1
from benzene-hexane to give 4b as pale white crystals (0.8 g, 76.2%), m.p. 112-115 °C; H-NMR: δ
7.47-7.42 (2H, m, Ph-H); 7.38-7.25 (4H, m, Ph-H × 3 and Ar-H); 6.88, (1H, d, J = 8.9 Hz, Ar-H); 5.10
13
(2H, s, CH2Ph); 3.89 (3H, s, OCH3). C-NMR: δ 171.0 (C), 152.2 (C), 145.9 (C), 136.7 (C), 130.6
(C), 128.4 (CH), 128.3 (CH), 128.2 (CH), 114.9 (CH), 108.7 (C), 76.0 (CH2), 56.2 (OCH3).
2-(2′-Benzyloxy-6′-bromo-3′-methoxybenzoyl)-1-methylene-6,7-methylenedioxy-1,2,3,4-tetrahydro-
isoquinoline (6a). A solution of acid 4b (3.6 g, 10.0 mmol) and thionyl chloride (3.9 g, 32.8 mmol) in
benzene (20 mL) was refluxed for 1 h. The solvent and excess thionyl chloride were removed under
vacuum to give acid chloride 4c as a yellow oil (3.7 g, 94.9%) which was used in the next step without
further purification. A solution of acid chloride 4c (1.9 g, 5.3 mmol) in dry benzene (20 mL) was
added dropwise over 10 min. to a solution of isoquinoline 5 [8] (1.0 g, 5.3 mmol) and triethylamine
(1.0 g) in dry benzene (20 mL), then the mixture was refluxed for 2 h. On cooling, the precipitated
triethylamine hydrochloride was filtered off. The filtrate was evaporated under vacuum to give
enamide 6a as a yellow oil (2.6 g, 84.4%) which was unstable and decomposed on standing. It was
1
immediately used in the next step without further purification. H-NMR: δ 7.38-7.23 (5H, m, Ph-H),
7.18(1H, d, J = 8.8 Hz, H-5′), 6.89 (1H, s, H-8), 6.75 (1H, d, J = 8.8 Hz, H-4′), 6.41 (1H, s, H-5), 5.90
(2H, AB q, J = 1.3 Hz, OCH2O), 5.14 (1H, d, J = 1.3 Hz, =CH2), 5.00 (2H, AB q, J = 10.8 Hz,
CH2Ph), 4.81 (1H, d, J = 1.3 Hz, =CH2), 4.13-4.02, 3.57-3.50 (2H, 2 m, CH2-3), 3.80 (3H, s, OCH3),
13
2.90-2.59 (2H, m, CH2-4); C-NMR: δ 165.0 (C), 152.1 (C), 147.8 (C), 146.5 (C), 145.3 (C), 141.4
(C), 137.4 (C), 134.3 (C), 129.0 (C), 128.4 (CH), 128.1 (CH), 127.9 (CH), 127.7 (CH), 125.1 (C),
113.4 (CH), 110.0 (C), 108.4 (CH), 104.4 (CH2), 103.8 (CH), 101.1 (CH2), 75.4 (CH2), 55.9 (OCH3),
41.6 (CH2), 28.8 (CH2).
(±)-Gusanlung A (1) and 9-benzyl-8-oxyberberrubine (8a). A solution of enamide 6a (2.7 g, 5.3
mmol), tributyltin hydride (3.4 g, 11.7 mmol) and 2,2′-azobis(isobutyronitrile) (0.2 g, 0.7 mmol) in dry
benzene (50 mL) was refluxed with stirring for 3 h., then the solvent was removed under vacuum. The
residue was washed with hexane (4 × 15 mL) and dissolved in chloroform (30 mL). The chloroform
layer was washed with brine, then dried over anhydrous sodium sulfate. The solvent was removed
under vacuum to give a yellow solid which was recrystallized from ethanol to give a 31.3% yield of a

Molecules 2009, 14
735
mixture of (±)-9-benzylgusanlung A (7a) and 9-benzyl-8-oxyberberrubine (8a) in a ratio of 78:22 from
1H-NMR analysis.
A solution of the mixture of 8a and 7a (303.7 mg, 0.7 mmol) in ethanol (50 mL) was hydrogenated
over 10% Pd/C (30.4 mg) at atmospheric pressure for 48 h. The catalyst was fittered off and the
solvent was removed under vacuum to give a crude yellow solid. Recrystallization of the crude solid
from ethanol gave (±)-gusanlung A (2) as a pale yellow soild (82.4 mg, 34.3%), m.p. 188-189 °C; UV
(MeOH) λmax nm (log ε): 219 (4.54), 271sh (3.87), 281 (3.98), 308 (4.16), 319 (4.15); IR νmax (film):
3737, 3650, 3585, 2919, 2852, 1748, 1634, 1615, 1581, 1542, 1506, 1488, 1456, 1386, 1356, 1336,
1315, 1262, 1239, 1154, 1084, 1069, 1037, 1001, 933, 858, 804, 792, 728 cm-1; MS (EI) m/z (%): 339
(M+, 55), 176 (100). 1H-NMR see Table 3, 13C-NMR and HMBC see Table 4.
A solution of iodine (4.6 g, 18.3 mmol) in dioxane (100 mL) was added dropwise over 30 min. to a
refluxing solution of the mixture of 7a and 8a (1.3 g, 3.0 mmol) and sodium acetate (1.5 g) in dioxane
(50 mL), then the mixture was refluxed for 6 h. On cooling, the sodium acetate was filtered off and the
precipitate was washed with chloroform (100 mL). The chloroform layer was washed with 5%
NaHSO3 (100 mL), dilute NH3 (30 mL), H2O (100 mL) then dried over anh. Na2SO4. Removal of the
solvent under vacuum gave a red solid which was recrystallized with ethanol to give 9-benzyl-8-
oxyberberrubine (8a) as red crystals (0.6 g, 50.0%), m.p. 190-192 °C. UV (MeOH) λmax nm (log ε):
206sh (4.62), 224 (6.31), 255sh (5.78), 312 (5.76), 342 (6.03), 369 (5.86), 387 (5.71); IR νmax (film):
2938, 2898, 2841, 1651, 1619, 1599, 1494, 1484, 1386, 1372, 1317, 1277, 1225, 1176, 1100, 1083,
1
939, 871, 834, 777, 734 cm-1; H-NMR: δ 7.73-7.68 (2H, m, Ph-H); 7.43-7.32 (3H, m, Ph-H); 7.32-
7.28 (2H, m, H-11 and H-12); 7.22 (1H, s, H-1), 6.72 (1H, s, H-13); 6.70 (1H, s, H-4); 6.00 (2H, s,
OCH2O); 5.16 (2H, s, CH2Ph); 4.31 (2H, t, J = 6.1 Hz, CH2-6); 3.88 (3H, s, OCH3); 2.88 (2H, t, J =
13
6.1 Hz, CH2-5); C-NMR: δ 160.2 (C), 151.7 (C), 148.4 (C), 148.2 (C), 147.3 (C), 138.1 (C), 135.6
(C), 132.4 (C), 130.1 (C), 128.7 (CH), 128.2 (CH), 127.7 (CH), 123.8 (C) , 122.5 (CH), 119.8 (C),
119.1 (CH), 107.9 (CH), 104.7 (CH), 101.4 (CH2), 101.3 (CH), 75.7(CH2), 56.9 (OCH3), 39.5 (CH2),
28.7 (CH2).
8-Oxyberberrubine (3). A solution of 8a (100.0 mg, 0.2 mmol) in ethanol (30 mL) and conc. HCl (30
mL) was refluxed for 3 h. On cooling, the solution was extracted with chloroform (50 mL). The extract
was washed with water (50 mL), then dried over anh. Na2SO4. Removal of the solvent under vacuum
gave a yellow solid which was recrystallized with ethanol to give 8-oxyberberrubine (3) as pale yellow
crystals (42.2 mg, 53.5%), m.p. 238-239 °C (Lit. [2] m.p. 240-241 °C); UV (MeOH) λmax nm (log ε):
225 (4.44), 258sh (3.99), 270 (3.87), 288 (3.69), 345 (4.16), 369 (4.13); IR νmax (film): 3011, 2893,
2836, 1645, 1594, 1490, 1393, 1320, 1267, 1228, 1181, 1087, 1033, 932, 826, 665 cm-1. 1H-NMR, 13C-
NMR and HMBC see Table 5.
2-(2′-Iodobenzoyl)-1-methylene-6, 7-methylenedioxy-1,2,3,4-tetrahydroisoquinoline (6b). A solution
of 2-iodobenzoyl chloride 4d (1.4 g, 5.4 mmol) in dry benzene (20 mL) was added dropwise over 10
min. to a solution of isoquinoline 5 [8] (1.0 g, 5.3 mmol) and triethylamine (1.0 g) in dry benzene (20
mL), then the mixture was refluxed for 2 h. On cooling, the precipitated triethylamine hydrochloride
was filtered off and the filtrate was evaporated under vacuum to give enamide 6b as a yellow oil (2.2
g, 99.1%) which was unstable and decomposed on standing, so it was immediately used in the next

Molecules 2009, 14
736
step without further purification. 1H-NMR: δ 8.07-6.84 (5H, m, Ar-H); 6.58 (1H, s, Ar-H); 5.92 (2H, s,
OCH2O); 5.18 (1H, br s, =CH2); 4.50 (1H, br s, =CH2); 4.12( 2H, br s, CH2); 2.95 (2H, br s, CH2); 13C-
NMR: δ 169.0 (C), 161.2 (C), 148.2 (C), 146.8 (C), 142.6 (C), 142.2 (CH), 139.3 (CH), 135.9 (C),
132.5 (C), 129.9 (CH), 128.3 (CH), 125.0 (C), 108.4 (CH), 106.2 (CH2), 103.9 (CH), 101.2 (CH2),
41.8 (CH2), 29.0 (CH2).
(±)-Gusanlung D (1) and 13,14-didehydrogusanlung D (8b). A solution of enamide 6b (2.9 g, 10.0
mmol) tributyltin hydride (11.7 g, 40.0 mmol) and 2,2′-azobis(isobutyronitrile) (1.6 g, 10.0 mmol) in
dry benzene (50 mL) was refluxed with stirring for 3 h., then the solvent was removed under vacuum.
The residue was washed with hexane (4 ×15 mL) and dissolved in chloroform (30 mL). The
chloroform layer was washed with brine, then dried over anhydrous sodium sulfate. The solvent was
removed under vacuum to give a solid which was recrystallized from ethanol to give a 39.0% yield of
a mixture of 1 and 8b in a ratio of 23:87 from 1H-NMR analysis.
A mixture of 1 and 8b (200.0 mg, 0.7 mmol), Pd/C (300.0 mg), hydrazine (50 mL), ethanol (50
mL) and ethyl acetate (50 mL) was refluxed for 48 h. The Pd/C was filtered and the filtrate extracted
with chloroform (80 mL). The extract was washed with 10% HCl (2 × 50 mL), water (50 mL) then
dried over anh. Na2SO4. Removal of the solvent under vacuum gave a yellow solid which was
recrystallized with ethanol to give pure (±)-gusanlung D (1) as pale yellow crystals (99.4 mg, 49.4%),
m.p. 175-176 °C (lit. [5] m.p. 175-177 °C). UV (MeOH) λmax nm (log ε): 206 (6.27), 230 (5.78), 253sh
(5.42), 290 (5.42), 335 (5.02), 365 (4.77); IR νmax (film): 2922, 1646, 1602, 1576, 1487, 1412, 1362,
1333, 1285, 1241, 1218, 1178, 1141, 1038, 936, 906, 853, 743, 636, 505 cm-1. 1H-NMR and 13C-NMR
see Tables 1 and 2.
Minimum inhibitory concentration (MIC)
MIC of (±)-gusanlung A (2) and (±)-gusanlung D (1) were determined by NCCLS microbroth
dilution methods [9]. (±)-Gusanlung A (2) and (±)-gusanlung D (1) were weighed and dissolved in
DMSO to make a solution of concentration 2.56 mg/mL. From this stock solution two-fold 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.
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Sample Availability: All stable products reported in this paper are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).