Potential cancer chemopreventive activity of simple isoquinolines, 1-benzylisoquinolines, and protoberberines

Article abstract of DOI:10.1016/j.phytochem.2005.10.007

Seventeen simple isoquinolines, 15 1-benzylisoquinolines, and 19 protoberberines were tested for their inhibitory activities against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol- 13-acetate (TPA) in Raji cells. Among the tested alkaloids, the inhibitory activity of all 1-benzylisoquinolines and 11 protoberberines was higher than that of β-carotene. The 1-benzylisoquinolines 19, 21, 22, 29, and 34 and protoberberines 41, 47-49, 51, 52, and 55 showed potent inhibitory effects on EBV-EA induction (96-100% inhibition at 1 × 10³ mol ratio/TPA). These alkaloids were more active than the naturally occurring alkaloids, 23, 25, 33, 53, and 54. In addition, fifteen simple isoquinolines, eighteen 1-benzylisoquinolines and eight protoberberines were evaluated with respect to their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. Nine simple isoquinolines, ten 1-benzylisoquinolines, and four protoberberines were more potent than α-tocopherol, and four 1-benzylisoquinolines, 20 and 28-30, exhibited potent activities (SC₅₀ 4.5-5.8 μM). Their activities were higher than the naturally occurring alkaloids, 23, 25, and 33. Therefore, some of the isoquinoline alkaloids indicating the high activity on both assays may be potentially valuable cancer chemopreventive agents. Structure-activity relationships are discussed for both tests.

Full text of DOI:10.1016/j.phytochem.2005.10.007

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 70–79
www.elsevier.com/locate/phytochem
Potential cancer chemopreventive activity of simple isoquinolines,
1-benzylisoquinolines, and protoberberines
a
a,
b
a
a
Wenhua Cui , Kinuko Iwasa *, Harukuni Tokuda , Akiko Kashihara , Yosuke Mitani ,
a
a
a
b
Tomoko Hasegawa , Yumi Nishiyama , Masataka Moriyasu , Hoyoku Nishino ,
c
c
d
Miyoji Hanaoka , Chisato Mukai , Kazuyoshi Takeda
a
Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan
Department of Biochemistry and Molecular Biology, Kyoto Prefectural University of Medicine, Kawaramachi-dori, kamigyo-ku, Kyoto 602-0841, Japan
b
c
Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology,
Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
d
Ebara Research Co. Ltd., 4-2-1, Honfujisawa, Fujisawa-shi 251-8502, Japan
Received 4 May 2005; received in revised form 10 September 2005
Available online 28 November 2005
Abstract
Seventeen simple isoquinolines, 15 1-benzylisoquinolines, and 19 protoberberines were tested for their inhibitory activities against
Epstein–Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Among
the tested alkaloids, the inhibitory activity of all 1-benzylisoquinolines and 11 protoberberines was higher than that of b-carotene.
The 1-benzylisoquinolines 19, 21, 22, 29, and 34 and protoberberines 41, 47–49, 51, 52, and 55 showed potent inhibitory effects on
EBV-EA induction (96–100% inhibition at 1 · 103 mol ratio/TPA). These alkaloids were more active than the naturally occurring
alkaloids, 23, 25, 33, 53, and 54. In addition, fifteen simple isoquinolines, eighteen 1-benzylisoquinolines and eight protoberberines were
evaluated with respect to their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. Nine simple isoquinolines, ten
1-benzylisoquinolines, and four protoberberines were more potent than a-tocopherol, and four 1-benzylisoquinolines, 20 and 28–30,
exhibited potent activities (SC50 4.5–5.8 lM). Their activities were higher than the naturally occurring alkaloids, 23, 25, and 33. There-
fore, some of the isoquinoline alkaloids indicating the high activity on both assays may be potentially valuable cancer chemopreventive
agents. Structure–activity relationships are discussed for both tests.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Isoquinoline alkaloids; Chemopreventive effects; Anti-tumor promoter; Epstein–Barr virus early antigen; Free radical scavenging activity;
Structure–activity relationships
1. Introduction
in the opium poppy, and many types of isoquinoline alka-
loids including protoberberines (Brossi, 1993; Szantay
et al., 1994).
Several plant and mammalian species contain simple
isoquinolines, such as salsolinol and its O-and N-methyl
analogues, 1-benzylisoquinolines, and protoberberines
(Lundstro¨m, 1983; Preininger, 1986; Brossi, 1993). The
1-benzylisoquinolines are related structurally to reticuline,
which is an intermediate in the biosynthesis of morphine
We have previously described the antimicrobial, antima-
larial, cytotoxic, and anti-HIV activities of isoquinoline
alkaloids (Iwasa et al., 2001a). Some of the tested com-
pounds proved to be significantly active in these assays.
The cytotoxic activities of several isoquinoline alkaloids
have already been reported (Iwasa et al., 2001a,b) and the
anti-tumor-promoting activities of some of isoquinoline-
type alkaloids have been published (Yasukawa et al., 1991;
*
Corresponding author. Tel.: +81 78 453 0031; fax: +81 78 435 2080.
E-mail address: k-iwasa@kobepharma-u.ac.jp (K. Iwasa).
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.10.007

W. Cui et al. / Phytochemistry 67 (2006) 70–79
71
Ito et al., 2001). The development of anti-tumor-promoters
has been regarded as the most effective method for the pre-
vention of carcinogenesis (Yamamoto et al., 1979; Verma
et al., 1980). The Epstein–Barr virus early antigen (EBV-
EA) activation assay has been considered to be an effective
indicator for the evaluation of anti-tumor-promoting activ-
ity (Ito et al., 1981). Compounds which inhibit EBV-EA
activation induced by the tumor promoter, 12-O-tetradeca-
noylphorbol-13-acetate (TPA), act as inhibitors of tumor
promotion in vivo (Konoshima et al., 1995). A series of
human illnesses such as cancer, diabetes, atherosclerosis,
etc., can be linked to the damaging action of reactive free
radicals (Konoshima et al., 1995). Thus, scavenging of free
radicals seems to play a considerable part in the pharmaco-
logical activity of antioxidative compounds.
To look for possible chemopreventive anti-tumor-pro-
moters, we carried out a primary screening of naturally
occurring isoquinolines [simple isoquinolines (1, 7 and 8),
1-benzylisoquinolines (23, 25, and 33), and protoberberines
(53, 54)] and related synthetic compounds using their inhib-
itory effects on the EBV-EA activation, induced by TPA,
as well as tests of their radical-scavenging activity against
1,1-diphenyl-2-picrylhydrazyl radical (DPPH). The struc-
ture–activity relationship is discussed for different alkyl
substituents on aliphatic or aromatic carbon atoms, the
position or number of the hydroxyl groups and the oxida-
tion state of the pyridine ring.
preparative HPLC. On the other hand, 1-benzylisoquino-
lines (28 and 29) were prepared by complete acid-catalyzed
ether cleavage of l-benzyl-5,6,30,40-tetramethoxyisoquino-
line (Iwasa et al., 2001a) and papaverine, respectively.
N-methylpapaveroline (34) was produced by the complete
acid-catalyzed ether cleavage of N-methylpapaverinium salt.
1-Benzyl-N-methylisoquinolinium salts 35 and 36 (Cassels
and Deulofeu, 1966) and protoberberinium salts, 37–40
(Iwasa et al., 1997), 41–50 (Iwasa et al., 1998a), and 53–55
and 60 (Iwasa et al., 1998b, 2003) had been prepared as
described. The 2,3,9,10- or 2,3,10,11-tetrahydroxyprotob-
erberines (51, 52, 57, and 58) were prepared by complete
acid-catalyzed ether cleavage of the corresponding 2,3,9,
10-or 2,3,10,11-tetramethoxy protoberberines. The struc-
tures of the synthetic compounds were determined by analy-
1
sis of their MS, HRMS, H NMR, and NOESY spectra.
Some compounds (1, 7, 8, 23, 25, 33, 53–56, 59 and 60) have
previously been identified from plant materials (Lundstro¨m,
1983; Preininger, 1986; Brossi, 1993; Claude et al., 1978;
Patra et al., 1987).
2.2. Inhibitory effects on EBV-EA induction
The inhibitory effects of seventeen simple isoquinolines
(1–11 and 13–18), fifteen 1-benzylisoquinolies (19, 21–27,
29–34 and 36), and nineteen protoberberines (37–55) on
EBV-EA activation induced by TPA were examined as a
primary screening of anti-tumor-promoting activities.
The inhibitory effects of 6,7-dihydroxytetrahydroiso-
quinolines, their N- or O-methyl derivatives, and their
3,4-dihydro and dehydro analogues bearing an 1-alkyl side
chain (1–11, 13–18) on TPA-induced EBV-EA activation,
their effects on the viability of Raji cells, and the 50% inhib-
itory concentration (IC50) values are shown in Table 1. All
tested compounds displayed the same or lower inhibition
(IC50 388–447) than that of the reference compound, b-car-
otene (IC50 400) that has been studied extensively in cancer
chemoprevention using animal models (Murakami et al.,
1996). The inhibitory activity of the tetrahydroisoquino-
lines, isoquinolines, and N-methylisoquinolinium salts
was stronger than that of dihydroisoquinolines (compare
1, 14, and 17 with 10). The inhibition decreased as the num-
ber of CH2 units in the side chain lengthened (1–4, 10–13,
and 14–16), thus suggesting that extension of the alkyl
function, and therefore relative lipophilicity of the C-1 sub-
stituent, may contribute to the inhibitory effect. There is
some increase or decrease of the inhibition by replacement
of the hydroxyl groups at C-6 and C-7 with dimethoxyl
groups (compare 6 with 7 and 10 with 9).
2. Results and discussion
2.1. Synthesis
The 6,7-dihydroxytetrahydroisoquinolines 1–5 had been
prepared previously (Iwasa et al., 2001a). 1-Methyl- (8),
1-ethyl-, 1-propyl-, l-butyl-6,7-dimethoxydihydroisoquino-
lines were prepared by Bischler–Napieralski cyclization of
the corresponding amides which were prepared from
3,4-dimethoxyphenylethylamine and the appropriate anhy-
drides (acetic, propionic, n-butyric, and valeric anhydrides).
The l-alkyl-6,7-dimethoxydihydroisoquinolines (e.g. 8) were
N-methylated to give the N-methyl derivatives (e.g. 9), and
acid-catalyzed ether cleavage gave the 6,7-dihydroxy-N-
methyldihydroisoquinolinium salts (10–13). Reduction of 9
afforded l,2-dimethyl-6,7-dimethoxytetrahydroisoquinoline
(7) which was converted to its 6,7-dihydroxy derivative (6).
The isoquinolines (14–18) had been prepared previously
(Iwasa et al., 2001a). Tetrahydropapaveroline (19) was
purchased. Commercially available papaverine was methyl-
ated to give N-methylpapaverinium salt which was reduced
by NaBH4 to give N-methyltetrahydropapaverine. N-Meth-
yltetrahydropapaveroline (20) was prepared by acid-cata-
lyzed ether cleavage of N-methyltetrahydropapaverine.
The 1-benzyl-N-methyltetrahydroisoquinolines (21–27) and
1-benzylisoquinoline (30–33) were prepared by partial
acid-catalyzed ether cleavage of N-methyltetrahydropapa-
verine and papaverine, respectively, and separation by
The inhibitory effects of the 1-benzyltetrahydroisoquino-
lines and their dehydro analogues on activation induced by
TPA and the viability of Raji cells were examined. The
results are shown in Table 2. All tested 1-benzylisoquinolines
displayed stronger inhibition (IC50 324–367) than b-carotene
(IC50 400). Among them, 19, 21, 22, 29, and 34 showed at
5 · 102 and 1 · 102 mol ratio/TPA higher inhibitory activity
than that of ginsenoside-Rg1 which is known as a strong

72
W. Cui et al. / Phytochemistry 67 (2006) 70–79
Table 1
Table 2
Inhibitory effects of simple isoquinoline alkaloids on TPA induced EBV-
EA activation (100%)a
Inhibitory effects of 1-benzylisoquinoline alkaloids on TPA induced EBV-
EA activation(100%)a
Compounds
Concentration (mol ratio/32 pmol
TPA)
IC50b(mol ratio/
32 pmol TPA)
Compounds
Concentration (mol ratio/
32 pmol TPA)
IC50b(mol ratio/
32 pmol TPA)
1000
500
100
10
1000
500
100
10
96.8
93.0
94.1
100
95.3
100
100
100
TIQ
1-BnTIQ
1
2
3
4
5
6
7
11.3 (60)
12.6 (60)
15.4 (60)
17.6 (60)
16.0 (60)
12.1 (60)
13.9 (60)
38.0
40.1
41.3
42.7
41.9
39.6
41.2
79.0
81.0
83.2
84.1
84.3
80.0
81.0
100
100
100
100
100
100
100
390
398
409
412
411
395
399
19
21
22
23
24
25
26
27
3.9(60)
4.0(60)
4.2(60)
6.2(60)
5.0(60)
7.7(60)
7.9(60)
7.0(60)
26.4
25.2
26.1
29.4
27.6
29.1
30.2
28.3
73.4
72.2
73.0
75.7
73.8
75.4
76.4
72.4
349
344
348
359
350
360
362
358
2HIQ
8
14.3 (60)
15.2 (60)
18.4 (60)
19.3 (60)
21.4 (50)
43.0
43.2
41.6
43.7
46.8
82.0
81.9
85.3
86.6
87.6
100
100
100
100
100
408
408
434
438
447
1-BnIQ
29
9
3.1(60)
6.3(60)
7.8(60)
8.3(60)
8.2(60)
2.5(60)
6.2 (60)
0 (80)
25.1
29.1
31.4
33.7
33.5
22.3
28.0
32.5
34.3
71.6
75.7
76.7
78.0
78.8
69.4
75.1
72.6
82.7
95.3
348
360
361
367
367
324
358
310
400
10
11
13
30
31
100
100
100
100
91.3
100
91.0
100
32
33
IQ
14
34
36
10.4 (60)
12.3 (60)
15.9 (60)
10.3 (60)
12.4 (60)
0 (80)
38.3
40.6
42.3
37.4
39.5
32.5
78.1
80.0
83.6
79.0
81.0
72.6
100
100
100
100
100
389
397
410
388
397
310
15
16
Ginsenoside-Rg1
b-carotene
9.1 (60)
17
18
a
Values represent percentages relative to the positive control value.
TPA (32 pmol, 20 ng) = 100%. Values in parentheses are viability per-
centages of Raji cell.
IC50 represents the mol ratio to TPA that inhibits 50% of positive
control (100%) activated with 32 pmol of TPA.
Ginsenoside
-Rg1
b-carotene
a
91.0
b
9.1 (60)
34.3
82.7
100
400
Values represent percentages relative to the positive control value.
TPA (32 pmol, 20 ng) = 100%. Values in parentheses are viability per-
centages of Raji cell.
From a structure–activity point of view, some features
can be pointed out. The inhibitory activity of 13-alkyl
and 8-alkyl-12-bromo derivatives decreased as the C-13
or C-8 alkyl chain was extended by one carbon (compare
37–39, and 42–44). Replacement of the methoxyl or hydro-
xyl groups on the aromatic rings with methylenedioxy
groups decreased the inhibitory activity (compare 39 with
40 and 53 with 54 and 55).
b
IC50 represents the mol ratio to TPA that inhibits 50% of positive
control (100%) activated with 32 pmol of TPA.
anti-tumor-promoter. Compound 34, bearing four hydroxyl
groups, displayed the strongest inhibition and its activity is
comparable to that of ginsenoside-Rg1.
From a structure–activity point of view, some trends
were observed. The inhibitory activity of the 1-benzyliso-
quinolines increased as the number of hydroxyl groups
on the aromatic ring increased (compare 21 or 22 with
23–27 and 29 with 30–33). The 1-benzyltetrahydroisoquin-
olines having more than three hydroxyl groups on the aro-
matic rings displayed significant inhibitory activity (19–22).
Among the 1-benzylisoquinolines (19, 29, and 34) bearing
four hydroxyl groups, the 1-benzyl-N-methylisoquinolin-
ium salt (34) showed the strongest inhibition, indicating a
contribution of quaternarization.
The inhibition by 2,3,9,10-oxygenated protoberberinium
salts (37–51) and by 2,3,10,11-oxygenated protoberberi-
nium salts (52–55) was examined (Table 3). Compounds
41–44, 46–52, and 55 displayed higher activity than b-car-
otene. The inhibitory activities at 5 · 102 and 1 · 102 mol
ratio/TPA of seven compounds (41–43, 48–50, and 55)
were comparable with ginsenoside-Rg1. The inhibitory
activities at 1 · 103, 5 · 102 and 1 · 102 mole ratio/TPA
of 51 and 52 bearing four hydroxyl groups were higher
than that of ginsenoside-Rg1.
On the basis of these results, it appears that in protober-
berinium salts, the size of substituents at the C-8 and C-13,
and type and position of the oxygenated substituents on
rings A and D influence the inhibitory activity. Replace-
ment of the methoxyl or hydroxyl groups by the methylen-
edioxy group on the rings A and D strongly influences the
inhibition.
1-Benzylisoquinolines 19, 21, 22, 29, and 34 and protob-
erberines 41, 47–49, 51, 52, and 55 showed potent inhibi-
tory effects on EBV-EA induction (96–100% inhibition at
1 · 103 mol ratio/TPA) as compared with those of the nat-
urally occurring alkaloids 23, 25, 33, 53, and 54. These syn-
thetic alkaloids might be valuable antitumor promoters.
They appear to be useful leads for further development
of potential cancer chemopreventive agents.
2.3. Free radical scavenging activity
Next, the ability of the isoquinoline-type alkaloids to
scavenge DPPH free radicals were examined. To evaluate
the free radical scavenging activity of the isoquinolines,

W. Cui et al. / Phytochemistry 67 (2006) 70–79
73
Table 4
Table 3
Radical scavenging activity of simple isoquinoline alkaloids
Inhibitory effects of protoberberine alkaloids on TPA induced EBV-EA
activation (100%)a
Alkaloids
SC50 (lM)a
Compounds
Concentration (mol ratio/
32 pmol TPA)
IC50b(mol ratio/
32 pmol TPA)
TIQ
1
2
3
4
5
6
7
14.2
16.0
13.8
19.6
15.8
1000
500
100
10
PB
37
9.5 (60)
10.1 (60)
12.9 (50)
9.7 (60)
2.5 (60)
5.0 (60)
5.9 (60)
7.0 (50)
10.6 (60)
8.3 (50)
2.7 (60)
2.0 (60)
2.4 (60)
4.8 (60)
0 (60)
31.8
34.5
36.3
32.3
23.1
27.4
28.0
29.6
34.9
30.2
25.9
23.8
24.6
26.8
22.7
21.8
37.6
31.7
24.5
32.5
34.3
74.7
77.7
79.0
75.3
73.1
71.5
72.7
74.8
78.5
75.0
74.3
73.5
72.8
73.5
71.5
69.3
80.3
77.8
71.4
72.6
82.7
100
100
100
100
93.1
96.0
97.4
100
100
100
361
364
382
363
327
350
353
359
381
363
346
325
343
352
304
300
394
363
326
310
400
38
39
26.9
>250
40
41
2HIQ
10
11
42
43
13.7
15.2
17.1
12.9
44
45
12
13
46
47
95.8
IQ
14
48
49
94.0
95.1
95.9
93.7
92.5
100
100
94.7
91.0
100
>250
>250
>250
>250
20.3
15
17
50
51
18
a-tocopherol
52
53
0 (60)
15.6 (60)
9.4 (60)
2.3 (60)
0 (80)
a
The compound concentration showing radical scavenging efficacy of
54
55
50% was defined as SC50
.
Ginsenoside-Rg1
b-carotene
analogues were examined. The data are shown in Table 5.
Among the tested alkaloids, 1-benzyltetrahydroisoquinoline
(19), 1-benzyl-N-methyltetrahydroisoquinolines (20–22),
and 1-benzylisoquinolines (28–31) bearing more than two
hydroxy groups displayed higher activity than a-tocopherol.
Among the 1-benzyl-N-methyltetrahydroisoquinolines, the
activity enhances as the number of the hydroxyl group on
the aromatic rings increases (compare 25 and 27 ! 23 and
24 ! 21 and 22 ! 20) (see Fig. 1).
9.1 (60)
a
Values represent percentages relative to the positive control value.
TPA (32 pmol, 20 ng) = 100%. Values in parentheses are viability per-
centages of Raji cell.
b
IC50 represents the molratio to TPA that inhibits 50% of positive
control (100%) activated with 32 pmol of TPA.
the concentration required to scavenge DPPH free radicals
by 50% (SC50) were determined. The antioxidant, a-
tocopherol was used as reference compound.
1-Alkylated tetrahydroisoquinolines, dihydroisoquino-
lines, isoquinolines, and their N-methyl analogues were
tested and the results are presented in Table 4. As shown
there, 6,7-dihydroxylated tetrahydroisoquinolines (1–5)
and the N-methyldihydroisoquinolines (10–13) displayed
higher radical-scavenging activity than that of a-tocoph-
erol. 6,7-Dimethoxytetrahydroisoquinoline (7), 6,7-dihydr-
oxylated isoquinolines (14 and 15) and their N-methyl
analogues (17 and 18) were inactive. Oxidation of tetrahy-
droisoquinoline to the dihydroisoquinolinium salt increa-
sed the activity (compare 6 with 10). Aromatization of
the ring B in the isoquinoline molecule led to disappearance
of the activity (compare 1 with 14, 2 with 15, 10 with 17,
and 11 with 18) as regardless of the presence of the hydro-
xyl groups at C-6 and C-7 which may contribute to increase
of the activity. The activity was decreased by N-methyla-
tion (compare 1 with 6). Replacement of the hydroxyl
groups by the methoxyl groups at C-6 and C-7 demolished
the activity (compare 6 with 7). The activity of tetrahydro-
isoquinolines and N-methyldihydroisoquinolines did not
always grow with increasing of the aliphatic chain at C-l
(compare 1–5 and 10–13).
Table 5
Radical scavenging activity of 1-benzylisoquinoline alkaloids
Alkaloids
SC50(lM)a
1-BnTIQ
19
20
21
22
23
24
25
26
27
7.4
4.5
7.0
7.2
21.9
17.7
31.3
23.7
27.7
1-BnIQ+
28
5.8
5.4
5.0
8.2
196
>250
15.4
>250
>250
20.6
29
30
31
32
33
34
35
36
a-tocopherol
a
The free radical scavenging activity of 1-benzyltetrahy-
droisoquinolines, 1-benzylisoquinolines, and their N-methyl
The compound concentration showing radical scavenging efficacy of
50% was defined as SC50
.

74
W. Cui et al. / Phytochemistry 67 (2006) 70–79
Fig. 1. DPPH radical scavenging activity of 1-benzyl-N-methyltetrahydroisoquinolines.
Table 6
In the 1-benzylisoquinolines, the activity decreased by
Radical scavenging activity of protoberberine alkaloids
N-methylation (compare 29 with 34). The activity grows
as the number of the hydroxyl groups increase (compare
30 with 31), and the 1-benzylisoquinolines and 1-benzyl-
N-methylisoquinolines having a hydroxyl group displayed
no activity (32, 33, 35, and 36). Shifting hydroxyl groups
at C-6 and C-7 to C-5 and C-6 had no influence on the
activity (compare 28 with 29).
Alkaloids
SC50 (lM)a
Protoberberines (PB)
51
52
55
56
12.3
14.1
>250
>250
Structure-radical scavenging activity relationship study
of 1-benzylisoquinolines pointed to the importance of the
number of the phenolic hydroxyl groups present for the
antiradical efficacy. It appears that the 1-benzylisoquino-
lines and their tetrahydro derivatives bearing more than
three hydroxyl groups on the aromatic rings are the most
effective radical scavengers except for the compounds with
quinolizidinium structure (Table 5).
Tetrahydroprotoberberines (TPB)
57
11.5
12.4
>250
>250
20.4
58
59
60
a-tocopherol
a
The compound concentration showing radical scavenging efficacy of
50% was defined as SC50
.
The free radical scavenging activity of 2,3,9,10- and
2,3,10,11-oxygenated protoberberinium salts (51, 52, 55,
56) and their tetrahydro analogues (57–60) were examined
(Table 6). Compounds 51, 52, 57, and 58 displayed higher
activity than that of a-tocopherol. In both the protober-
berinium salts and their tetrahydro derivatives, 2,3,9,10-
oxygenated alkaloids showed slightly higher activity as
compared with that of their 2,3,10,11-oxygenated ana-
logues. Tetramethoxy derivatives (55, 56, 59, and 60) are
inactive. The phenolic hydroxyl groups on the aromatic
rings A and D influence the activity strongly.
Among the isoquinoline alkaloids tested, some 1-benzyl-
isoquinolines (SC50 4.5–5.8 lM for 20, 28–30, SC50 7.0–
8.2 lM for 19, 21, 22, 31) displayed higher free radical
scavenging activity than that of the simple isoquinolines
(SC50 12.9–14.2 lM for 1, 3, 10, and 13) and the protob-
erberines (SC50 11.5–14.1 lM for 51, 52, 57, and 58). These
alkaloids were more active than naturally occurring alka-
loids 23 (SC50 21.9 lM), 33, 56, and 59 (SC50 > 250lM),
and the reference a-tocopherol (SC50 20.3–20.6 lM). These
compounds possessing free radical scavenging activity
might be applied to the prevention of carcinogenesis.
Among the three types of isoquinoline alkaloids, 1-ben-
zylisoqinolines indicated the highest activity in both assays.
There appears to be a correlation between free radical scav-
enging and tumor promoter-inhibitory activities within the
1-benzylisoquinolines from the point of view that the activ-
ities in both assays depend mainly on the number of hydro-
xyl groups on the aromatic rings. The high activity of the
benzylisoquinolines may be explained by the ability to
donate a hydrogen atom from the hydroxyl groups and
thereby scavenge free radicals.
Of the test compounds prepared on the basis of the
natural products, the synthetic compounds in both assays
displayed higher activity in comparison with the correspond-
ing natural products. The results of the present investigation
suggest that some 1-benzylisoquinoline and protoberberine
alkaloids might be useful leads for the further development
of potential cancer chemopreventive agents. A study exam-

W. Cui et al. / Phytochemistry 67 (2006) 70–79
75
ining the tumor-promoting inhibitory activity of some com-
pounds in vivo is in progress.
(3H, s, 6-OCH3), 4.00 (2H, t, J = 7.5 Hz, H-3), 4.01 (3H,
s, 7-OCH3), 6.83 (1H, s, H-5), 7.22 (1H, s, H-8); SIMS
m/z [M ꢀ Cl]+ 220; HRMS m/z [M ꢀ Cl]+ 220.1347
(C13H18NO2 requires 220.1336).
3. Experimental
To a solution of the methiodide 9 (3 g) in MeOH (50 ml)
was added, in portions, NaBH4 (1 g), and the mixture was
stirred for 2 h. To the mixture was added H2O (30 ml). The
solution was concentrated and extracted with CHCl3. The
extract was dried (Na2SO4) and evaporated under reduced
pressure. The residue was dissolved in MeOH (50 ml) and
conc. HCl (0.5 ml), and the solvent was evaporated in vacuo
to give carnegine hydrochloride 7 (2.04 g, 97%): m.p. 210–
211 ꢁC (lit. Brown et al., 1972, m.p. 210–211 ꢁC); 1H NMR
(CD3OD, 500 MHz) d 1.66 (3H, d, J = 7.0 Hz, CH3), 2.95
(3H, s, N–CH3), 3.08 (2H, m, H-4), 3.42 and 3.64 (each
1H, m, H-3), 4.48 (1H, q, J = 7.0 Hz, H-1), 3.82 (6H, s, 6-
and 7-OCH3), 6.78 (1H, s, H-8), 6.80 (1H, s, H-5); SIMS
m/z [M ꢀ Cl]+ 222; HRMS m/z [M + H ꢀ Cl]+ 222.1500
(C13H20NO2 requires 222.1493). Carnegine hydrochloride
7 (100 mg) was heated in 47% HBr (1 ml) at 125ꢁ for
20 min. The solvent was evaporated under reduced pressure,
and the residue was purified by preparative HPLC [H2O
(0.05% TFA)/MeOH(0.05% TFA)] and converted to the
amorphous 6–HCl (70 mg, 58%): 1H NMR (CD3OD,
500 MHz) d 1.61 (3H, d, J = 7.0 Hz, CH3), 2.93 (3H, s, N–
CH3), 2.99 (2H, m, H-3), 3.39 and 3.61 (each 1H, m, H-4),
4.41 (1H, q, J = 7.0 Hz, H-1), 6.60 (1H, s, H-5 or H-8) and
6.61 (1H, s, H-5 or H-8); SIMS m/z [M ꢀ Cl]+ 194; HRMS
m/z [M + H ꢀ Cl]+ 194.1189 (C11H16NO2 requires
194.1180).
3.1. General
Mps: uncorr. Conventional 1H NMR and NOESY spec-
tra were obtained on a Varian VXR-500S spectrometer
(1H: 500 MHz) using CD3OD as the solvent, if not stated
otherwise, with TMS as internal standard. Mass spectra
were determined on a Hitachi M-4100 instrument at
75 eV. SIMS (glycerol). LC was performed on a Cosmosil
5 C18-AR (4.6 i.d. · 150 mm or 20 i.d. · 250 mm) reversed
phase column. The mobile phase was 0.1 M NH4OAc
(0.05% trifluoroacetic acid, TFA, A), to which MeOH
(0.05% TFA, B) was added by a linear gradient. The flow
rate was 1 ml/min; detection: 280 nm.
3,4-Dimethoxyphenylethylamine, tetrahydropapavero-
line hydrochloride (19) and palmatine chloride (56) were
obtained from commercial suppliers. The isoquinolines 1–5
and isoquinolines 14–18 had been prepared previously
(Iwasa et al., 2001a). 1-Benzyl-N-methylisoquinolines 35
and 36 had been prepared previously according to Cassels
and Deulofeu, 1966. Protoberberinium salts, 37–40 (Iwasa
et al., 1997), 41–50 (Iwasa et al., 1998a), and 53–55 and 60
(Iwasa et al., 1998b, 2003), had been prepared previously.
3.2. Synthesis of isoquinolines 6–9
A mixture of 3,4-dimethoxyphenylethylamine hydro-
chloride (20 g) and Ac2O (15 ml) was allowed to react at
room temperature for 2–3 h. Then ice water was added
and the mixture was stirred overnight. The solution was
extracted with CHCl3, the solvent was removed at reduced
pressure and the resulting crystals were washed with Et2O
to give the appropriate acetamide (21.7 g, Yield: 88%),
m.p. 85–90 ꢁC. A solution of this amide (5 g) in dry ben-
zene (50 ml) and POCl3 (4 ml) was heated to reflux for
2 h and then allowed to stand at room temperature. The
resultant crystals were purified to give 8 (6.89 g, Yield:
99%, m.p. 132–133 ꢁC) as a phosphate (H3PO4) which
was suspended in CHCl3 and treated with NH4OH solu-
tion. The CHCl3 extracts were dried and evaporated to
afford the free base. The resulting free base (100 mg) was
dissolved in MeOH–HCl, and the solvent was removed at
reduced pressure to give 8 hydrochloride (m.p. 214–
215 ꢁC). The free base obtained from 8 phosphate
(770 mg) was dissolved in a mixture of MeOH and Me2CO
(1:1) (20 ml), and after addition of CH3I (1 ml) the mixture
was refluxed at room temperature for 30 min. The resulting
crystals were recrystallized from MeOH to give 9 iodide
(640 mg, 75%, m.p. 169–171 ꢁC), which was converted to
3.3. Preparation of 1-alkyl-6,7-dihydroxy-N-
methylisoquinolinium salts 10–13
Dihydroisoquinolinium salt 9 (100 mg) was heated in
47% HBr (1 ml) at 120–123 ꢁC for 2 h. The solvent was
evaporated under reduced pressure and the residue was
crystallized from acetone to give 10 bromide (88 mg,
73%):
m.p.
191–197 ꢁC
(dec.);
1H
NMR
(CDCl3 + CF3COOD, 500 MHz)d 2.75 (3H, s, CH3), 3.07
(2H, t, J = 7.5 Hz, H-4), 3.71 (3H, s, N–CH3), 3.94 (2H,
t, J = 7.5 Hz, H-3), 6.91 (1H, s, H-5), 7.56 (1H, s, H-8);
SIMS m/z [M ꢀ Br]+ 192; HRMS m/z [M ꢀ Br]+
192.1024 (C11H14NO2 requires 192.1024).
The N-methylisoquinolinium salts 11–13 were prepared
similarly to the synthesis of 10 starting from 3,4-dimeth-
oxyphenethylamine and the appropriate anhydrides (propi-
onic, n-butyric, and valeric anhydrides). 11 bromide
(57 mg, 72%): m.p. 200–201 ꢁC; 1H NMR (CD3OD,
500 MHz) d 1.34 (3H, t, J = 7.5 Hz, CH2CH3), 3.02 (2H,
t, J = 7.5 Hz, H-4), 3.10 (2H, q, J = 7.5 Hz, CH2CH3),
3.71 (3H, s, N–CH3), 3.94 (2H, t, J = 7.5 Hz, H-3), 6.79
(1H, s, H-5), 7.36 (1H, s, H-8); SIMS m/z [M ꢀ Br]+ 206;
HRMS 206.1180 (C12H16NO2 requires 206.1178); 12 bro-
1
the
chloride,
m.p.
79–80 ꢁC;
1H
NMR
mide (71 mg, 89%): m.p. 200–203 ꢁC; H NMR (CD3OD,
(CDCl3 + CF3COOD, 500 MHz) d 2.79 (3H, s, CH3),
3.13 (2H, t, J = 7.5 Hz, H-4), 3.74 (3H, s, N–CH3), 3.94
500 MHz) d 1.13 (3H, t, J = 7.5 Hz, CH2CH2CH3), 1.72
(2H, m, CH2CH2CH3), 3.01 (2H, t, J = 7.5 Hz, H-4), 3.05

76
W. Cui et al. / Phytochemistry 67 (2006) 70–79
(2H, m, CH2CH2CH3), 3.71 (3H, s, N–CH3), 3.94 (2H, t,
J = 7.5 Hz, H-3), 6.78 (1H, s, H-5), 7.35 (1H, s, H-8); SIMS
m/z [M ꢀ Br]+ 220; HRMS 220.1351 (C13H18NO2 requires
H-4), 9.05 (1H, d, J = 7.0 Hz, H-3); SIMS m/z [M ꢀ Cl]+
354; HRMS m/z [M ꢀ Cl]+ 354.1710 (C21H24NO4 requires
354.1704).
1
220.1336); 13 bromide (65 mg, 80%): m.p. 201–204 ꢁC; H
To a solution of N-methylpapaverinium chloride
NMR (CD3OD, 500 MHz) d 1.03 [3H, t, J = 7.5 Hz,
(CH2)3CH3], 1.56 (2H, m, CH2CH2CH2CH3), 1.67 (2H,
m, CH2CH2CH2CH3), 3.01 (2H, t, J = 7.5 Hz, H-4), 3.07
(2H, t, J = 7.5 Hz, CH2CH2CH2CH3), 3.70 (3H, s, N–
CH3), 3.94 (2H, t, J = 7.5 Hz, H-3), 6.78 (1H, s, H-5),
7.35 (1H, s, H-8); SIMS m/z [M ꢀ Br]+ 234; HRMS
234.1493 (C14H20NO2 requires 234.1502).
(519 mg) in MeOH (200 ml) was added NaBH4 (1 g), and
the mixture was stirred at room temperature for 2 h. After
dilution with water (30 ml), the mixture was concentrated
and extracted with CHCl3. The CHCl3 extracts were washed
with water, dried, and evaporated to give crystals which were
recrystallized from MeOH–Me2CO to give N-methyltetra-
hydropapaverine (200 mg, 42%): mp. 113–113.5ꢁ; 1H
NMR (CDCl3, 500 MHz) d 2.54 (3H, s, N–CH3), 2.56–
3.20 (4H, m, H-3 and -4), 2.77 (1H, dd,J = 13.5,7.7 Hz,
H-9), 3.14 (1H, dd, J = 13.5, 5.0 Hz, H-9), 3.57 (3H, s,
7-OCH3), 3.70 (1H, dd, J = 7.7, 5.0 Hz, H-1), 3.79 (3H, s,
30-OCH3), 3.84 (3H, s, 6-OCH3), 3.85 (3H, s, 40-OCH3),
6.06 (1H, s, H-8), 6.56 (1H, s, H-5), 6.60 (1H, d,
J = 2.0 Hz, H-20), 6.64 (1H, dd, J = 8.0, 2.0 Hz, H-60), 6.77
(1H, d, J = 8.0 Hz, H-50); SIMS m/z [M + H]+ 358; HRMS
358.2021 (C21H28NO4 requires 358.2017).
A solution of N-methyltetrahydropapaverine (130 mg) in
47% HBr (1 ml) was refluxed for 30 min. The solvent was
evaporated in vacuo. The crystalline product was recrystal-
lized from MeOH–Me2CO to give N-methyltetrahydropap-
averoline hydrobromide 20 (46 mg, 33%): m.p. 226–228 ꢁC
1
(dec.); H NMR (DMSO–d6–D2O, 500 MHz) d 2.77 (3H,
s, N–CH3), 2.8–3.6 (6H, m, H-3, ꢀ4 and ꢀ9), 4.38 (1H, br
s, H-1), 6.13 (1H, s, H-8), 6.47 (1H, dd, J = 8.0, 2.0 Hz, H-
60), 6.60 (1H, d, J = 2.0 Hz, H-20), 6.61 (1H, s, H-5), 6.70
(1H, d, J = 8.0 Hz, H-50); SIMS m/z [M + H ꢀ Br]+ 302;
HRMS 302.1395 (C17H20NO4 requires 302.1391).
3.5. Preparation of phenolic 1-benzyl-N-
methyltetrahydroisoquinolines (21)–(27)
The mixtures of the phenolic compounds prepared by
the acid-catalyzed partial ether cleavage of 1-benzyl-N-
methyltetrahydropapaverine were separated by preparative
HPLC [(A) 0.1 M NH4OAc (0.05% TFA)/(B) MeOH
(0.05% TFA) initial A/B 85/15, 85 min 0/100] to give 21–
27. The 1H NMR and MS spectroscopic data of these com-
pounds will be presented elsewhere.
3.4. Preparation of N-methyltetrahydropapaveroline (20)
A solution of commercially available papaverine hydro-
chloride (Sigma, 5 g) and CH3I (5 ml) in MeOH (40 ml)
and CHCl3 (20 ml) was placed in a glass-stoppered bottle
and heated for 13 h at 110 ꢁC in a oil bath. The solvent
was evaporated in vacuo, the residue was dissolved in
CHCl3 and washed with 5% Na2S2O3. The CHCl3 solution
was dried (Na2SO4) and evaporated in vacuo. The residue
(iodide) was converted to the chloride (4.03 g, 78%) by
treatment with AgCl in MeOH. N-methylpapaverinium
chloride: m.p. 145–158 ꢁC (dec.); 1H NMR (CDCl3,
500 MHz) d 3.82 (3H, s, 6- or 7-OCH3), 3.84 (3H, s, 6-
or 7-OCH3), 4.01 (3H, s, 30- or 40-OCH3), 4.15 (3H, s, 30-
or 40-OCH3), 4.65 (3H, s, N–CH3), 5.07 (2H, s, H-9),
6.27 (1H, dd, J = 8.5, 2.0 Hz, H-60), 6.71 (1H, d,
J = 8.5 Hz, H-50), 6.88 (1H, d, J = 2.0 Hz, H-20), 7.50
(1H, s, H-5), 7.53 (1H, s, H-8), 8.19 (1H, d, J = 7.0 Hz,
3.6. Preparation of 5,6,30,40-tetrahydroxy-1-
benzylisoquinoline (28)
Benzyl-5,6,30,40-tetramethoxyisoquinoline
hydrobro-
mide (Iwasa et al., 2001a) (100 mg) in 47% HBr (1 ml)
was refluxed for 3 h. The solution was evaporated in vacuo.
The residue was crystallized from acetone to give 28 hyd-
robromide (80 mg, 74%): m.p. 223–237 ꢁC; 1H NMR
(CD3OD, 500 MHz) d 4.68 (2H, s, H-9), 6.59 (1H, dd,
J = 8.0, 1.5 Hz, H-60), 6.66 (1H, br d, H-20), 6.73 (1H, d,
J = 8.0 Hz, H-50), 7.55 (1H, d, J = 9.0 Hz, H-8), 8.10
(1H, d, J = 9.0 Hz, H-7), 8.12 (1H, d, J = 6.5 Hz, H-3),
8.33 (1H, d, J = 6.5 Hz, H-4); SIMS m/z [M ꢀ Br]+ 284;
HRMS 284.0916 (C16H14NO4 requires 284.0921).

W. Cui et al. / Phytochemistry 67 (2006) 70–79
77
3.7. Preparation of papaveroline (29)
solvent was evaporated in vacuo. The crystalline product
was recrystallized from MeOH–Me2CO to give 34 bro-
mide (240 mg, 64%): m.p. 236–242 ꢁC (dec.); 1H NMR
(DMSO–d6, 500 MHz) d 4.21 (3H, s, N–CH3), 4.67
(2H, s, H-9), 6.34 (1H, dd, J = 8.0, 2.0 Hz, H-60), 6.44
(1H, d, J = 2.0 Hz, H-20), 6.67 (1H, d, J = 8.0 Hz, H-
50), 7.44 (1H, s, H-5), 7.74 (1H, s, H-8), 8.09 (1H, d,
J = 6.5 Hz, H-3), 8.36 (1H, d, J = 6.5 Hz, H-4); SIMS
m/z [M ꢀ Br]+ 298; HRMS 298.1074 (C17H16NO4
requires 298.1079).
Papaverine hydrochloride (4 g) in 47% HBr (40 ml) was
refluxed for 55 h. The resulting crystals were recrystallized
from MeOH to give papaveroline hydrobromide 29 (3.27 g,
84%), m.p. 255–260 ꢁC (dec.) which was converted to the
trifluoroacetate by HPLC with H2O–MeOH (0.05%
1
TFA) as the mobile phase. H NMR (CDCl3, 500 MHz)
d 4.63 (2H, s, H-9), 6.62 (1H, dd, J = 8.0, 2.0 Hz, H-60),
6.72 (1H, d, J = 2.0 Hz, H-20), 6.75 (1H, d, J = 8.0 Hz,
H-50), 7.44 (1H, s, H-5), 7.76 (1H, d, J = 1.0 Hz, H-8),
7.96 (1H, br d, J = 6.5 Hz, H-4), 8.11 (1H, d, J = 6.5 Hz,
H-3); SIMS m/z [M ꢀ Br]+ 284; HRMS 284.0936
(C16H14NO4 requires 284.0921).
3.10. Preparation of protobererinium salts 51 and 52
Palmatine (Iwasa et al., 1997) and Pseudopalmatine
(Iwasa et al., 2001a) in 47% HBr were refluxed for several
hours to give their tetrahydroxy derivatives [51 bromide:
m.p. 239–251 ꢁC (dec.) and 52 bromide: m.p. 254–261 ꢁC
(dec.)] in quantitative yield. The bromides were converted
to the trifluoroacetates by HPLC [H2O (0.05% TFA)/MeOH
3.8. Preparation of l-benzylisoquinolines (30–33)
The mixtures of the phenolic compounds prepared by
the acid-catalyzed partial ether cleavage of papaverine were
separated by the preparative HPLC [(A) 0.1 M NH4OAc
(0.05% TFA)/(B) MeOH (0.05% TFA) initial A/B 75/25,
1
(0.05% TFA)]. 51 trifluoroacetate: H NMR (DMSO-d6,
500 MHz) d 3.15 (2H, t, J = 6.5, H-5), 4.83 (2H, t, J = 6.5,
H-6), 6.81 (1H, s, H-4), 7.47 (1H, s, H-1), 7.57 (1H, d,
J = 8.5 Hz, H-12), 7.71 (1H, d, J = 8.5 Hz, H-11), 8.41
(1H, s, H-13), 9.64 (1H, s, H-8); SIMS m/z [M ꢀ CF3COO]+
296; HRMS 296.0911 (C17H14NO4 requires 296.0921); 52
trifluoroacetate: 1H NMR (DMSO-d6, 500 MHz) d 3.12
(2H, t, J = 6.5 Hz, H2-5), 4.69 (2H, t, J = 6.5 Hz, H2-6),
6.80 (1H, s, H-4), 7.35 (1H, s, H-12), 7.42 (1H, s, H-9), 7.45
(1H, s, H-1), 8.23 (1H, s, H-13), 9.11 (1H, s, H-8); SIMS
1
10 min 50/50, 20 min 80/20] to give 30–33. The H NMR
and MS spectroscopic data of these compounds will be pre-
sented elsewhere.
3.9. Preparation of N-methylpapaveroline (34)
A
solution of N-methylpapaverinium chloride
(300 mg) in 47% HBr (3 ml) was refluxed for 8 h. The

78
W. Cui et al. / Phytochemistry 67 (2006) 70–79
m/z [M ꢀ CF3COO]+ 296; HRMS 296.0931 (C17H14NO4
(1H, m, H-5), 3.52 (1H, m, H-6), 3.62 (1H, m, H-13), 3.84
(1H, m, H-6), 4.32 (1H, d, J = 15.5 Hz, H-8), 4.64 (1H, dd,
J = 12.0, 4.5 Hz, H-13a), 4.70 (1H, d, J = 15.5 Hz, H-8),
6.65 (1H, s, H-4), 6.66 (1H, d, J = 8.0 Hz, H-12), 6.78 (1H,
s, H-1), 6.79 (1H, d, J = 8.0 Hz, H-11); SIMS m/z
[M ꢀ CF3COO]+ 300; HRMS 300.1232 (C17H18NO4
requires 300.1235); 58 trifluoroacetate, 1H NMR (CD3OD,
500 MHz) d 2.92 (1H, m, H-5), 2.99 (1H, dd, J = 17.0,
12.0 Hz, H- 13), 3.18 (1H, m, H-5), 3.47 (1H, m, H-6), 3.61
(1H, m, H-13), 3.78 (1H, m, H-6), 4.43 (2H, br s, H-8), 4.65
(1H, dd, J = 12.0, 4.5 Hz, H-13a), 6.61 (1H, s, H-5), 6.64
(1H, s, H-4), 6.70 (1H, s, H-12), 6.78 (1H, s, H-1); SIMS
m/z [M ꢀ CF3COO]+ 300; HRMS 300.1225 (C17H18NO4
requires 300.1235).
requires 296.0921).
3.11. Preparation of tetrahydroprotoberberines 57 and 58
Tetrahydropseudopalmatine (Iwasa et al., 2003) and tet-
rahydropalmatine in 47% HBr were refluxed for several
hours to give their tetrahydroxy derivatives [57 hydrobro-
mide: m.p. 248–256 ꢁC (dec.) and 58 hydrobromide: m.p.
258–268 ꢁC (dec.)] in quantitative yield.
The hydrobromides were converted to the trifluoroace-
tates by HPLC [H2O (0.05% TFA)/MeOH (0.05% TFA)].
1
57 trifluoroacetate, H NMR (CD3OD, 500 MHz) d 2.96
(1H, m, H-5), 3.02 (1H, dd, J = 17.0, 12.0 Hz, H-13), 3.18

W. Cui et al. / Phytochemistry 67 (2006) 70–79
79
Cassels, B.K., Deulofeu, V., 1966. Resolution of O-benzoyl-( )-codamine.
Tetrahedron (Suppl. 8, Part II), 485–490.
3.12. In vitro EBV-EA activation experiment (Takemura
et al., 1995)
Claude, M., Edouard, S., Jean-Claude, R., 1978. Analyseconformationnelle
de pseudoprotoberberines naturelles. Org. Magn. Reson. 11, 398–400.
Ito, Y., Yanase, S., Fujita, J., Harayama, T., Takashima, T., Imanaka, H.,
1981. A short term in vitro assay for promoter substances using human
lymphoblastoid cells. Cancer Lett. 13, 29–37.
Ito, C., Itoigawa, M., Tokuda, H., Kuchide, M., Nishino, H., Furukawa,
H., 2001. Chemopreventive activity of isoquinoline alkaloids from
Corydalis plants. Planta Med. 67, 473–475.
Iwasa, K., Kamigauchi, M., Sugiura, M., Nanba, H., 1997. Antimicrobial
activity of some 13-alkyl substituted protoberberinium salts. Planta
Med. 63, 196–198.
Iwasa, K., Lee, D.-U., Kang, S.-I., Wiegrebe, W., 1998a. Antimicrobial
activity of 8- alkyl- and 8-phenyl-substituted berberines and their 12-
bromo derivatives. J. Nat. Prod. 61, 1150–1153.
Iwasa, K., Nanba, H., Lee, D.-U., Kang, S.-I., 1998b. Structure–activity
relationships of protoberberines having antimicrobial activity. Planta
Med. 64, 748–751.
Iwasa, K., Moriyasu, M., Tachibana, Y., Kim, H.-S., Wataya, Y.,
Wiegrebe, W., Bastow, K.F., Cosentino, L.M., Kozuka, M., Lee,
K.H., 2001a. Simple isoquinoline and benzylisoquinoline alkaloids as
potenial antimicrobial, antimalarial, cytotoxic, and anti-HIV agents.
Bioorg. Med. Chem. 9, 2871–2884.
Iwasa, K., Moriyasu, M., Yamori, T., Turuo, T., Lee, D.-U., Wiegrebe,
W., 2001b. In vitro cytotoxicity of the protoberberine-type alkaloids. J.
Nat. Prod. 64, 896–898.
Iwasa, K., Kuribayashi, A., Sugiura, M., Moriyasu, M., Lee, D.-U.,
Wiegrebe, W., 2003. LC-NMR and LC-MS analysis of 2, 3, 10, 11-
oxygenated protoberberine metabolites in Corydalis cell cultures.
Phytochemistry 64, 1229–1238.
The inhibition of EBV-EA activation was assayed using
Raji cells (virus non-producer) which were cultivated in 8%
FBS RPMI 1640 medium. The indicator Raji cells
(1 · 106 cells/ml) were incubated at 37 ꢁC for 48 h in 1 ml
of the medium containing n-butyric acid (4 mM, inducer),
32 pmol of TPA (20 ng/ml in DMSO), and 32, 16, 3.2, and
0.32 nmol of the test compound (added as a DMSO solu-
tion). Smears were made from the cell suspension. The acti-
vated cells were stained by high-titer EBV-EA positive sera
from nasopharyngeal carcinoma (NPC) patients and
detected by a conventional indirect immunofluorescence
technique. In each assay, at least 500 cells were counted,
and the experiments were repeated three times. The average
extent of EA induction was compared with that of positive
control experiments with n-butyric acid (4 mM) plus TPA
(32 pmol) in which EA induction was ordinarily around
40%. In this screening method, the cell viability required
for the judgment of inhibitory effects was more than 60%.
3.13. Determination of the scavenging effect on DPPH
radicals (Blois, 1958)
Ethanol (100 ll) was added to individual wells of a 96-well
plate. The test compounds were dissolved in DMSO and
diluted with EtOH to adjust to 500 lM concentration. The
final solvent concentration was 0.25% DMSO (v/v). The
sample solution (100 ll) was added to individual wells of a
96-well plate by the twofold dilution, and EtOH solution
(100 ll) of DPPH radical (final concentration was 100 lM)
was also added. The final concentration of the test com-
pounds was from 0.24 to 250 lM. A control sample contain-
ing EtOH solution (100 ll) of DPPH radical and EtOH
(100 ll) was prepared in the 96-well plate. The 96-well plate
was incubated at 25ꢁ for 30 min in the dark. After incuba-
tion, the absorbance decrease was determined by measuring
the optical density change at 550 nm with a microplate lumi-
nescence reader Lucy 2 (ALOKA). The radical-scavenging
activity expressed as % inhibition against DPPH radical,
was calculated according to Yen and Duh (1994): % Inhibi-
tion = [(AB ꢀ AA)/AB] · 100, (AA is the absorbance of the
tested sample after 30 min; AB is the absorbance of blank
sample). The data presented are the average from two or
three independent experiments.
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