Simple isoquinoline and benzylisoquinoline alkaloids as potential antimicrobial, antimalarial, cytotoxic, and anti-HIV agents

Article abstract of DOI:10.1016/S0968-0896(01)00154-7

Twenty-six simple isoquinolines and 21 benzylisoquinolines were tested for antimicrobial, antimalarial, cytotoxic, and anti-HIV activities. Some simple isoquinoline alkaloids were significantly active in each assay, and may be useful as lead compounds for developing potential chemotherapeutic agents. These compounds include 13 (antimicrobial), 25, 26, and 42 (antimalarial), 13 and 25 (cytotoxic), and 28 and 29 (anti-HIV). A quaternary nitrogen atom of isoquinolium or dihydroisoquinolinium type may contribute to enhanced potency in the first three types of activities. In contrast, anti-HIV activity was found with tetrahydroisoquinoline and 6,7-dihydroxyisoquinolium salts.

Full text of DOI:10.1016/S0968-0896(01)00154-7

Bioorganic & Medicinal Chemistry 9 (2001) 2871–2884
Simple Isoquinoline and Benzylisoquinoline Alkaloids as Potential
Antimicrobial, Antimalarial, Cytotoxic, and Anti-HIV Agents
Kinuko Iwasa,a Masataka Moriyasu,a Yoko Tachibana,b Hye-Sook Kim,c
b
e
Yusuke Wataya,c Wolfgang Wiegrebe,d Kenneth F.Bastow, L.Mark Cosentino,
Mutsuo Kozukab and Kuo-Hsiung Leeb,*
aKobe Pharmaceutical University, 4-19-1 Motoyamakita, Higashinada-ku, Kobe 658-8558, Japan
bNatural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7360, USA
cFaculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan
dInstitute of Pharmacy, Regensburg University, D-93040 Regensburg, Germany
eBiotech Research Laboratories, Inc., 217 Perry Parkway, Gaithersburg, MD 20877, USA
Received 19 January 2001; accepted 4 May 2001
Abstract—Twenty-six simple isoquinolines and 21 benzylisoquinolines were tested for antimicrobial, antimalarial, cytotoxic, and
anti-HIV activities.Some simple isoquinoline alkaloids were significantly active in each assay, and may be useful as lead com-
pounds for developing potential chemotherapeutic agents.These compounds include 13 (antimicrobial), 25, 26, and 42 (anti-
malarial), 13 and 25 (cytotoxic), and 28 and 29 (anti-HIV).A quaternary nitrogen atom of isoquinolium or dihydroisoquinolinium
type may contribute to enhanced potency in the first three types of activities.In contrast, anti-HIV activity was found with tetra-
hydroisoquinoline and 6,7-dihydroxyisoquinolium salts. # 2001 Elsevier Science Ltd.All rights reserved.
Introduction
including benzyltetrahydroisoquinolines.Based on these
reports, we investigated SARs based on different alkyl
substituents at the C-1 and N-2 positions and on the
6,7-OH groups as well as the oxidation state of the
pyridine ring.
Several plant and mammalian species contain simple
1,2,3,4-tetrahydroisoquinolines, such as salsolinol and
its O- and N-methyl analogues, and 1-benzyltetrahy-
droisoquinolines.1,2 The latter compounds are related
structurally to reticuline, which is an intermediate in
biosynthesis of morphine in the opium poppy and
also may play a role in the biosynthesis of mammalian
morphines.2,3
In addition, prompted by a report of the anti-HIV
activity of michellamine B, a naphthylisoquinoline
alkaloid dimer from Ancistrocladus korupenis,11 we
investigated and describe herein the anti-HIV activities
of tetrahydroisoquinolines, 3,4-dihydroisoquinolines,
isoquinolines, 1- and 3-benzylisoquinolines, and corre-
sponding quaternary salts.
We have previously described the antimalarial activity
of structurally related protoberberine alkaloids4,5 and
were interested in further examining simple isoquinoline
and benzylisoquinoline alkaloids and their aromatized
derivatives for antimalarial activity.Also, prior litera-
ture reports have described the neurotoxicity of the
tetrahydroisoquinoline salsolinol6 and antitumor struc-
ture–activity relationship (SAR) patterns7 of isoquino-
line derivatives, and numerous reports have described
the antimicrobial activity of isoquinoline alkaloids,8ꢀ10
Our focus in this study was to identify lead compounds
and find preliminary SAR trends.Analogue synthesis
based on these initial results and mechanism of action
studies will be presented in future papers.
Chemistry
1-Methyl-6,7-dihydroxytetrahydroisoquinoline, salsoli-
nol (1), was prepared previously.12 1-Ethyl-, 1-propyl-,
1-isopropyl-, and 1-pentyl-6,7-dihydroxytetrahydro-
*Corresponding author.Fax: +1-919-966-3893; e-mail: khlee@email.
unc.edu
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd.All rights reserved.
PII: S0968-0896(01)00154-7

2872
K. Iwasa et al. / Bioorg. Med. Chem. 9 (2001) 2871–2884
isoquinolines (2–5) were prepared by condensation of
dopamine with an appropriate aldehyde (Scheme 1).
Treating 3 with propyl iodide gave the O,N-tripropyl
derivative (6), which was converted to its 3,4-dihydro
quaternary salt (7) by oxidation using iodine.Similarly,
treating 1–5 with benzyl chloride produced O,N-tri-
benzyl derivatives 8–12.Oxidation of 10 by iodine gave
dehydro analogues were tested against two Gram-posi-
tive (Staphylococcus aureus, Bacillus subtilis) bacteria,
two Gram-negative (Salmonella enteritidis, Escherichia
coli), and one fungus (Candida albicans) by the liquid
dilution method.Papaverine ( 30) and its analogues (31–
50) also were tested.The minimum inhibitory con-
centrations (MIC) are presented in Table 4
O,N-tribenzyl-1-propyl-3,4-dihydroisoquinoline
(13).
Dehydrogenation of 8–12 with Pd-C in refluxing tetralin
gave the 6,7-dihydroxy compounds 14–18.Compounds
15–18 were O-propylated to give 19–22. N-Methyla-
tion of 19 and 20 forded the isoquinolinium salts 23
and 24, respectively.The 6,7-dihydroxy isoquinolines 14
and 15 were O-benzylated, then N-methylated to pro-
duce the corresponding salts 25 and 26, respectively,
which were debenzylated by reflux in 20% HCl to give
the 6,7-dihydroxy salts (27 and 28, respectively).6,7-
Dimethoxy-3,4-dihydroisoquinoline (29) was prepared
according to literature methods.13 Papaverine (30) was
commercially available, and its analogues (31–39 and
41) were prepared by previously reported methods.14ꢀ23
1-Benzyl- (30–38) and 3-benzyl- (39) isoquinolines were
N-methylated to give 40 and 42–50.The structures of 2–
29 (Scheme 1) and 42–50 (Chart 1) were confirmed by
1H NMR data (Tables 1 and 2) , including NOESY, and
LSIMS and HRLSIMS data (Table 3).
All tetrahydroisoquinolines (1–6, 8, 10–12) and iso-
quinolines (14–16, 18, 20–22) displayed low activity
(>500 mg/ml) against all tested strains.Oxidation of
6,7-dipropyloxy-N-propyl tetrahydroisoquinoline (6) to
its dihydroisoquinolinium salt (7) increased activity
slightly in some strains.While N-methylation of 6,7-
dihydroxyisoquinolines 14 and 15 to the corresponding
dihydroxyisoquinolinium salts 27 and 28 did not affect
activity, N-methylation of 6,7-dipropyloxyisoquinolines
19 and 20 to the isoquinolinium derivatives 23 and 24
increased activity slightly.Thus, from these data, both
N-quaternization/alkylation and O-alkylation appear to
be important to enhanced activity.Accordingly, com-
pounds 13, 25, and 26, which contain both a N-alky-
lated quaternary nitrogen of the dihydroisoquinolinium
or isoquinolinium-type and O-benzyl groups on the 6,7-
hydroxy groups, displayed significant antimicrobial
activity.Activity of the latter two 6,7-dibenzyloxy- N-
methyl salts increased as the C-1 alkyl chain lengthened
by one carbon (compare 26 with 25).Among the tested
simple isoquinolines, 6,7-dibenzyloxy-N-benzyl-1-pro-
pyl-dihydroisoquinolinium chloride (13) exhibited the
highest activity against all strains tested, and its activity
was higher than that of kanamycin sulfate. O,N-Benzy-
lation was more effective than O,N-propylation (com-
pare 13 with 7 and 25, 26 with 23, 24).Compound 13
Results and Discussion
Antimicrobial activity
6,7-Dihydroxytetrahydroisoquinolines, their N- and O-
alkyl or -benzyl derivatives, and their 3,4-dihydro and
Scheme 1.

K. Iwasa et al. / Bioorg. Med. Chem. 9 (2001) 2871–2884
2873
appears to be a useful new lead for further development
of antimicrobial agents.
by only 0–31%.The corresponding aromatic iso-
quinolines (14–16) and N-methylated derivatives (27
and 28) showed slight inhibitory activity (EC50 ranged
from 1.8E-05 to 5.9E-06) and low selectivity indexes
(0.7–1.3). As found with antimicrobial activity, the O,N-
tribenzyl-dihydroisoquinolinium salt (13) displayed
more inhibitory activity than the O,N-tripropyl salt (7);
however, both selectivity indexes were low.6,7-Dipro-
pyloxy-N-methyl isoquinolinium salts (23 and 24)
inhibited P. falciparum with EC50 values on the order of
10ꢀ7 molar, but again, their selectivity indexes were low.
However, the 6,7-dibenzyloxy-N-methyl salts (25 and
26) were potent antimalarial agents with higher selec-
tivity indexes compared with the other test compounds.
In conclusion, a quaternary nitrogen atom, especially in
an isoquinolinium rather than a dihydroisoquinolinium
ion, contributed to increased antimalarial activity.Ben-
zylation of the C-6 and C-7 hydroxyl groups increased
activity significantly (compare 25 and 26 with 27 and 28,
respectively), and to a larger extent than propylation of
these groups (compare 26 and 13 with 23 and 7, respec-
tively).Among the tested simple isoquinolines, 6,7-
dibenzyloxy-N-methyl isoquinolinium salts (25 and 26)
showed the most potent antimalarial activity.
Papaverine (30) and related compounds (31–50) were
tested against two Gram-positive, two Gram-negative,
and one fungal microorganisms.Benzylisoquinolines
(30–39) with a hydrogen substituent on the nitrogen atom
showed weak activity against all test strains (Table 4).
Among N-methylated benzylisoquinolinium salts (40–
50), only 48 and 49 displayed slightly increased activity
against S. aureus, B. subtilis, and S. enteritidis.Thus,
activity increased when the four methoxy groups of N-
methylpapaverine (40) were replaced by ethoxy groups
(48).
Antimalarial activity
Sixteen simple isoquinolines (1–5, 7, 13–16, and 23–28)
and 12 benzylisoquinolines (30, 40–50) were tested in
vitro against human malaria Plasmodium falciparum
FCR-3.The antimalarial activity of each compound
was determined as percentage reduction.The compound
concentration required to inhibit cell growth by 50%
was expressed as EC50.To evaluate compounds’ toxicity
for mammalian cells, the concentration causing a 50%
growth reduction (IC50) of mouse mammary FM3A
Among the tested benzylisoquinolines, 30, 40, 41, 43–48
and 50 displayed slight inhibitory activity and their
selectivity indexes were low.Only compounds 42 and 49
inhibited P. falciparum with EC50 values in the order of
10ꢀ7 molar and their selectivity indexes increased com-
pared with other tested compounds.Introducing a methyl
group into the C-3 or C-9 position increased activity
(compare 42 and 49 with 40 and 48, respectively), while
cells, a model of the host, was determined.The IC
/
50
EC50 ratios for the compounds were calculated as
selectivity indexes, and these ratios were used as an
evaluation of antimalarial activity.The results are pre-
sented in Table 5.Among the simple isoquinolines, 6,7-
dihydroxytetrahydroisoquinolines (1–5) with varying C-1
alkyl side chains inhibited the growth of P. falciparum
Chart 1.

Table 1. 1H NMRa data of simple isoquinolines 2–29
Compd
1-H
3-H
4-H
5-H
6-H
2b
4.29 dd 1H (J=8, 4.5)
3.51 ddd 1H (J=12, 7, 6)
3.31 ddd 1H (J=12, 7.5, 6)
3.50 ddd 1H (J=12, 7, 6)
3.31 ddd 1H (J=12.5, 7.5, 6)
2.99 ddd 1H (J=17.5, 7.5, 6)
2.90 ddd 1H (J=17.5, 7, 6)
2.98 ddd 1H (J=17.5, 7.5, 6)
2.90 ddd 1H(J=17.5, 7.5, 6)
6.67 s 1H
6.61 s 1H
3b
4.34 dd 1H (J=8, 5)
4.29 d 1H (J=5)
6.65 s 1H
6.66 s 1H
6.61 s 1H
6.62 s 1H
4b
3.53 ddd 1H (J=12.5, 6, 4.5)
3.25 ddd 1H (J=12.5, 10, 5.5)
2.98 ddd 1H (J=17.5, 7, 6)
2.85 ddd 1H (J=17.5, 5.5, 4.5)
5b
6c
4.33 dd 1H (J=8, 5)
4.07 dd 1H (J=9, 4)
3.49 ddd 1H (J=12.5, 7, 6)
3.30 ddd 1H (J=12.5, 7.5, 6)
3.70 m 1H
2.98 ddd 1H (J=17, 7.5, 6)
2.90 ddd 1H (J=17, 7, 6)
2.97d m 2H
6.65 s 1H
6.66 s 1H
6.61 s 1H
6.51 s 1H
3.51 m 1H
7c
8c
4.01 t 2H (J=7.5)
3.61 m 1H
3.37 m 1H
3.08 t 2H (J=7.5)
3.04 m 1H
6.81 s 1H
6.75 s 1H
7.19 s 1H
6.53 s 1H
4.28 q 1H (J=6.5)
3.95 dd 1H (J=10, 4)
3.79 d 1H (J=8)
2.89 dd 1H (J=17.5, 5.5)
3.04 m 1H
2.93 dd 1H (J=18, 6.5)
3.16 m 1H
3.07 m 1H
10c
11b
12c
3.66 m 1H
3.46 m 1H
3.81 m 1H
3.45 m 1H
3.67 m 1H
3.04 m 1H
6.79 s 1H
6.61 s 1H
6.79 s 1H
6.42 s 1H
7.00 s 1H
6.46 s 1H
3.93 dd 1H (J=9, 3.5)
3.46 m 1H
2.93 dd 1H (J=18, 7)
3.25 t 2H (J=7.5)
7.88 d 1H (J=6.8)
7.90 d 1H (J=6.8)
7.90 d 1H (J=6.5)
7.86 d 1H (J=6.5)
7.74 d 1H (J=6.5)
7.84 d 1H (J=6.5)
7.73 d 1H (J=6.5)
7.90 d 1H (J=6.5)
7.92 d 1H (J=6.5)
8.00 d 1H (J=6.8)
7.80 d 1H (J=6.8)
7.85 d 1H (J=6.8)
7.86 d 1H (J=7.3)
3.16 t 2H (J=7.5)
13c
14b
15b
16b
18b
20c
21c
22c
23c
24c
25b
26b
27b
28b
29b
4.20 t 2H (J=7.5)
8.03 d 1H (J=6.8)
8.05 d 1H (J=6.8)
8.05 d 1H (J=6.5)
8.03 d 1H (J=6.5)
8.36 d 1H (J=6.5)
8.65 d 1H (J=6.5)
8.33 d 1H (J=6.5)
8.44 d 1H (J=6.5)
8.57 d 1H (J=6.5)
8.30 d 1H (J=6.8)
8.27 d 1H (J=6.8)
8.17 d 1H (J=6.8)
8.15 d 1H (J=7.3)
3.91 t 2H (J=7.5)
6.88 s 1H
7.63 s 1H
7.69 s 1H
7.68 s 1H
7.65 s 1H
7.21 s 1H
7.27 s 1H
7.20 s 1H
7.25 s 1H
7.25 s 1H
7.69 s 1H
7.69 s 1H
7.72 s 1H
7.71 s 1H
7.10 s 1H
7.24 s 1H
7.37 s 1H
7.40 s 1H
7.89 s 1H
7.37 s 1H
7.38 s 1H
7.49 s 1H
7.38 s 1H
7.40 s 1H
7.37 s 1H
7.88 s 1H
7.77 s 1H
7.34 s 1H
7.36 s 1H
7.38 d 1 H (J=3.5)
8.73 d 1H (J=3.5)
(continued)

Table 1 (continued)
Compd
6,7-O-Alkyl substituent
C-1 Substituent
N-2 Substituent
2b
3b
4b
5b
6c
7c
1.11 t 3H (J=7.5); 1.92 dq 1H (J=8, 7.5);
2.11 qd 1H (J=7.5, 4.5)
1.05 t 3H (J=7.5); 1.53 m 2H; 1.86 m 1H;
2.01 m 1H
0.92 and 1.45 d each 3H (J=7);
2.45 m 1H
0.95 t 3H (J=7); 1.40 m 4H; 1.50 m 2H;
1.86 and 2.10 m each 1H
1.05e and 1.06e t 3H (J=7.5); 1.84d m 4H;
3.93 m 4H
0.93e t 3H (J=7); 1.48 and 1.69 m each 1H;
2.17 m 2H
0.96 t 3H (J=7); 1.84d m 2H; 2.97d m 2H
1.06e and 1.07e t 3H (J=7.5); 1.90 m 4H; 3.97
and 3.98 t 2H (J=6.5)
1.08e t 3H (J=7.5); 1.73 m 2H; 3.04 m 2H
1.12e t 3H (J=7.5); 1.85e m 2H; 4.09 t 2H (J=6.5)
8c
10c
5.14 and 5.16 s each 2H; 7.3–7.5d m 10H
5.15 and 5.22 d each 1H (J=13); 5.19 s 2H
7.3–7.5d m 10H
1.57 d 3H (J=6.5)
0.75 t 3H (J=7); 1.02 and 1.17 and 1.52 and
2.01 m each 1H
4.27 and 4.22 d each 1H (J=12.5); 7.3–7.5d
4.23 and 4.14 d each 1H (J=13); 7.3–7.5d
m
m
11b
12c
5.14 and 5.16 d each 1H (J=12.5); 5.18 s 2H;
7.26 –7.41d m 10H
5.15 and 5.19 d each 1H (J=12.5); 5.18 s 2H;
7.3–7.5d m 10H
0.63 and 0.93 d each 3H (J=7); 2.03 m 1H
4.40 and 4.13 d each 1H (J=13); 7.44–7.52 m 5H
4.23 and 4.13 d each 1H (J=13); 7.3–7.5d m 5H
0.80 t 3H (J=7); 1.04–1.28 m 6H; 1.55
and 2.07 m each 1H
13c
14b
15b
16b
18b
20c
5.27 and 5.31 s each 2H; 7.3–7.5d m 10H
0.96 t 3H (J=7); 1.54 m 2H; 3.02 s 3H
3.02 s 3H
5.45 s 2H; 7.3–7.5d m 5H
1.49 t 3H (J=7.5); 3.40 q 2H (J=7.5)
1.03 t 3H (J=7.5); 1.91 m 2H; 3.35 t 2H (J=7.5)
0.94 t 3H (J=7); 1.43 m 4H; 1.85 m 2H; 3.33 m 2H
1.06 t 3H (J=7.5); 1.98d m 2H; 3.47 t 2H
(J=7.5)
1.13 and 1.14 t each 3H (J=7.5); 1.98d m 4H;
4.16 and 4.20 t each 2H (J=6.5)
21c
12c
23c
24c
1.13 and 1.14 t each 3H (J=7.5); 1.99 m 4H;
4.17 and 4.22 t each 2H (J=6.5)
1.13 and 1.14 t each 3H (J=7.5); 1.98 and 1.99 m
each 2H; 4.14 and 4.20 t each 2H (J=6.5)
1.12 and 1.13 t each 3H (J=7.5); 1.98 m 4H;
4.16 and 4.21 t each 2H (J=6.5)
1.12 and 1.14 t each 3H (J=7.5); 1.98 m 4H;
4.15 and 4.21 t each 2H (J=6.5)
5.43 s 4H; 7.32–7.42 m 6H; 7.52–7.56 m 4H
5.44 and 5.47 s each 2H; 7.31–7.43 m 6H;
7.52–7.56 m 4H
1.78 d 6H (J=7); 4.07 m 1H
0.93 t 3H (J=7.5); 1.18 and 1.37 and 1.43 m
each 2H; 3.46 t 2H (J=8)
1.48 t 3H (J=7.5); 3.51 q 2H (J=7.5)
4.46 s 3H
4.48 s 3H
1.21 t 3H; 1.84 m 2 H; 3.43 t 2H (J=8)
25b
26b
3.10 s 3H
1.33 t 3H (J=7.5); 3.54 q 2H (J=7.5)
4.33 s 3H
4.36 s 3H
27b
28b
29b
3.05 s 3H
1.44 t 3H (J=7.5); 3.49 q 2H (J=7.5)
4.30 s 3H
4.34 s 3H
3.89 and 3.99 s each 3H
aCoupling constants (Hz in parentheses).
bThe solvent was CD3OD.
cThe solvent was CDCl3.
dOverlap with other protons.
eAssignments may be interchanged.

Table 2. 1H NMRa Data of benzylisoquinolines 31–50
Compd
2-Me
3-H or 1-H or 3-Me
4-H
5-H or 7-H
7.36 s 1H
8-H
9-H
20-H
50-H
60-H,–CH2OH or–COMe
31
8.43 d 1H
7.41 d 1H
(J=5.5)
7.50 d 1H
(J=5.5)
8.20 d 1H
(J=5.5)
2.64c s 3H
8.36 d 1H
(J=6.0)
8.28 d 1H
(J=7.0)
8.27 d 1H
(J=6.0)
2.65c s 3H
7.03 s 1H
(J=5.5)
7.09b s 1H
(J=5.5)
7.37 d 1H
(J=5.5)
7.26 s 1H
7.42 d 1H
(J=6.0)
8.22 d 1H
(J=7.0)
7.75 d 1H
(J=6.0)
7.23 s 1H
4.84 q 1H
6.81 d 1H
(J=6.5)
6.76 d 1H
(J=3.0)
6.97 brs1H
6.86 dd 1H
(J=8.0)
(J=8.0, 3.0)
6.76 brs 2H
32
33
8.45 d 1H
7.25b s 1H
7.09 s 1H
7.67 s 1H
4.57 s 2H
6.76 s 1H
6.94 s 1H
4.74e s 2H
34
35
6.95 s 1H
7.05 s 1H
7.55 s 1H
7.31 s 1H
4.94 s 2H
4.50 s 2H
6.76 s1H
6.71 d 1H
(J=1.5)
6.73 brd 1H
(J=1.0)
7.11 d 1H
(J=2.0)
6.81 d 1H
(J=2.0)
7.08 d 1H
(J=2.0)
6.88 d 1H
(J=2.0)
6.46 s 1H
7.22 s 1H
6.71 d 1H
(J=8.0)
6.72 d 1H
(J=8.0)
6.76 d 1H
(J=8.0)
6.74 d 1H
(J=8.5)
6.86 d 1H
(J=8.0)
6.71 d 1H
(J=8.5)
7.37b s 1H
2.65f s 3H
6.77 dd 1H
(J=8.0, 1.5)
6.95 dd 1H
(J=8.0, 1.0)
6.83 dd 1H
(J=8.0, 2.0)
6.72 dd 1H
(J=8.5, 2.0)
6.94 dd 1H
(J=8.0, 2.0)
6.27 dd 1H
(J=8.5, 2.0)
2.58f s 3H
36
37
38
39
40
41
42
43
44
7.70g d 1H
(J=9.5)
7.18 s 1H
8.42 d 1H
(J=9.5)
7.50 s 1H
4.98 s 2H
4.89 s 2H
4.47 s 2H
4.52 s 2H
5.07 s 2H
5.36 s 2H
6.93 s 1H
7.10 s 1H
7.50b s 1H
7.44b s 1H
7.41 s 1H
7.26 s 1H
7.46b s 1H
7.53b s 1H
7.49b s 1H
7.28 s 1H
7.30 s 1H
9.25J s 1H
7.48b s 1H
4.65 s 3H
4.64 s 3H
4.84 s 3H
4.42 s 3H
4.50 s 3H
9.05 d 1H
(J=7.0)
9.23 d 1H
(J=7.0)
9.45 d 1H
(J=7.0)
9.53 d 1H
8.19 d 1H
(J=7.0)
8.21d 1H
(J=7.0)
8.20 d 1H
(J=7.0)
8.45 d 1H
(J=7.0)
8.11 d 1H
5.38 q 1H
(J=7.5)
6.66 d 1H
(J=1.5)
7.00 d 1H
(J=2.0)
6.97 s1H
6.87 d 1H
(J=8.5)
6.75 d 1H
(J=8.5)
5.72 s 1H
6.81 dd 1H
(J=8.5, 1.5)
6.31 dd 1H
(J=8.5, 2.0)
5.24e s 2H
7.68 s 1H
(J=7.0)
7.57b s 1H
(J=7.0)
7.46 s 1H
7.51b s 1H
8.62 d 1H
7.46b s 1H
(J=7.0)
7.33 s 1H
7.48b s 1H
4.85 s 2H
45
46
4.39 s 3H
4.62 s 3H
3.03c s 3H
9.05 d 1H
(J=6.5)
8.85 d 1H
(J=7.0)
9.00 d 1H
(J=7.0)
2.86c s 3H
8.16 s 1H
8.18 d 1H
(J=6.5)
8.33 d 1H
(J=7.0)
8.09 d 1H
(J=7.0)
7.91 s 1H
5.42 s 2H
5.02 s 2H
6.64 s1H
6.53 d 1H
(J=2.0)
6.93 brd 1H
(J=2.0)
6.78 d 1H
(J=2.0)
6.90 d 1H
(J=2.0)
7.35 s 1H
6.71 d 1H
(J=8.0)
6.68 d 1H
(J=8.5)
6.72 d 1H
(J=8.5)
6.72 d 1H
(J=8.5)
2.56f s 3H
6.46 dd 1H
(J=8.0, 2.0)
6.22 dd 1H
(J=8.5, 2.0)
6.29 dd 1H
(J=8.5, 2.0)
6.33 dd 1H
(J=8.5, 2.0)
6.70 dd 1H
(J=8.5, 2.0)
47
48
49
50
4.67 s 3H
4.61 s 3H
4.39 s 3H
4.61 s 3H
7.71g d 1H
(J=9.0)
7.39b s 1H
8.32 d 1H
(J=9.0)
7.44b s 1H
5.18 s 2H
4.96 s 2H
5.13 s 2H
4.38 s 2H
7.28 s 1H
7.09s 1H
7.41 s 1H
8.00 s 1H
10.92J s 1H
7.50 s 1H
6.79 d 1H
(J=2.0)
6.88 d 1H
(J=8.5)
(continued)

Table 2 (continued)
Compd
–OMe or–OEt at
and
OMe or OEt or OCH2O at
and
Me or¼CH2 at
C-6
C-7 or C-5
3.76 s 3H
C-30
C-40
C-9
31
32
3.99 s 3H
4.03 s 3H
3.81 s 3H
3.88 s 3H
1.82c d 3H
(J=6.5)
5.99d s 1H
5.50d s 1H
3.85 s 3H
3.78 s 3H
3.80 s 3H
33
34
35
36
37
38
39
40
41
42
4.05 s 3H
3.98 s 3H
4.00 s 3H
4.11 s 3H
3.88 s 3H
3.91 s 3H
3.73 s 3H
3.64 s 3H
3.87 s 3H
3.89 s 3H
5.88 s 2H
4.13 s 3H
4.03h s 3H
3.90 s 3H
3.78 s 3H
4.06 qi 2H, 1.43 ti 3H
4.01 qi 2H, 1.39 ti 3H
3.90 s 3H
4.30 qi 2H,1.57 ti 3H
4.19 qi 2H,1.52 ti 3H
4.08 s 3H
4.16 qi 2H, 1.51 ti3H
4.04 qi 2H, 1.42 ti3H
4.08 s 3H
4.01 qi 2H, 1.39 ti 3H
3.97 qi 2H, 1.36 ti 3H
3.88 s 3H
4.15 s 3H
4.13 s 3H
4.10 s 3H
4.01 s 3H
3.84 s 3H
3.65 s 3H
3.82b s 3H
3.70 s 3H
3.77 s 3H
3.84b s 3H
3.94 s 3H
3.87 s 3H
1.20c d 3H
(J=7.5)
6.46d s 1H
5.61d s 1H
43
4.19 s 3H
3.91 s 3H
3.88 s 3H
3.90 s 3H
44
45
46
47
48
49
50
4.12 s 3H
4.13 s 3H
4.15 s 3H
3.96 s 3H
3.83 s 3H
4.01 s 3H
3.41 s 3H
3.77 s 3H
3.85 s 3H
3.94 s 3H
5.93 s 2H
4.15 s 3H
4.07h s 3H
3.85 s 3H
4.02 qi 2H, 1.41 ti 3H
4.04 qi 2H, 1.41 ti 3H
3.87 s 3H
3.81 s 3H
4.01 qi 2H, 1.42 ti 3H
4.02 qi 2H, 1.42 ti 3H
3.91 s 3H
4.36 qi 2H,1.60 ti 3H
4.36 qi 2H,1.59 ti 3H
4.08b s 3H
4.17 qi 2H, 1.51 ti3H
4.17 qi 2H, 1.51 ti3H
4.09b s 3H
aCoupling constants (Hz in parentheses), the solvent was CDCl3.
bAssignments may be interchanged.
cMe.
d
¼CH2.
eCH2OH.
fCOMe.
g7-H.
hC-5.
iCoupling constant (J=7.0 Hz)1-H.

2878
K. Iwasa et al. / Bioorg. Med. Chem. 9 (2001) 2871–2884
introducing a substituent such as acetyl or hydroxy-
methyl into the C-60 position decreased activity (com-
pare 41 or 44 with 40).
or showed marginal activity only in the HCT-8 cell line
(14, 15, 27).
6,7-Dimethoxydihydroisoquinolium chloride (29) was
inactive, while 1-ethyl-6,7-dipropyloxy-N-methylisoqui-
nolinium chloride (23) was active against KB and IA9
cells.The 6,7, N-tribenzylated dihydroisoquinolinium
salt (13) was active against five cell lines and had an
ED50 of 4.4 mg/mL in U-87 MG cells.6,7-Dibenzyloxy-
1-methyl-N-methyl isoquinolinium chloride 25 showed
broad spectrum activity against all six cell lines tested,
with ED50 values < 1.0 mg/mL in five cell lines and >1
mg/mL in HCT-8 cells.Generally, the trends found
above in the antimicrobial and antimalarial assays were
also present in the cytotoxicity assays. N-alkylation to
give a dihydroisoquinolium- or isoquinolium-type salt
combined with O-alkylation of the 6,7-hydroxy groups
led to the most potent compounds.Benzylation of the
C-6 and C-7 hydroxy groups significantly increased
cytotoxicity (compare 25 with 27), whereas propylation
had a weaker effect (compare 23 with 28).
Cytotoxicity evaluation
Fourteen simple isoquinolines (1–5, 13–16, 23, 25, 27–
29) and 21 benzylisoquinolines (30–50) were assayed for
in vitro cytotoxicity against six human tumor cell lines,
including lung carcinoma (A-549), ileocecal carcinoma
(HCT-8), ovarian cancer (1A9), breast cancer (MCF-7),
glioblastoma (U-87 MG), and epidermoid carcinoma
(KB).The cytotoxicity data are given as an ED value
50
for each cell line, the concentration of compound that
causes a 50% reduction in adsorbance at 562 nm rela-
tive to untreated cells using the SRB assay, and are
shown in Table 6.
All tetrahydroisoquinolines (1–5) tested were inactive in
all cell lines.6,7-Dihydroxyisoquinolines and 6,7-dihy-
droxyisoquinolium salts were either inactive (16 and 28)
Table 3. Physical, mass spectral, and HPLC analysis data of isoquinolines and benzylisoquinolines
Compd
Mp
(ꢁC) (dec)
Recryst solvent
Yield
Formula
LSIMS m/z
[MꢀCl]+
HR-LSIMS
HPLC (CR, min)a
[MꢀCF3COO]+
Calcd
Found
Ib,c,d
IIb,c,d
2
3
4
5
6
7
8
213–219
223–233
215–225
164–170
Amorph
Amorph
Amorph
Amorph
Amorph
Amorph
179–181
268–284
206–217
194–206
Amorph
101–102
127–128
Amorph
73–76
MeOH
MeOH
MeOH
Et2O
69
92
85
90
41
C11H16NO2
C12H18NO2
C12H18NO2
194
208
208
236
334
332
450
478
478
506
476
176
190
204
232
288
288
316
288
302
370
384
190
204
192
368
366
384
410
338
354
410
424
354
194.1199
208.1343
208.1337
236.1641
333.2627
332.2603
450.2454
478.2731
478.2756
506.3056
476.2590
176.0732
190.0880
204.1024
232.1324
288.1985
288.1976
316.2298
288.1960
302.2143
370.1808
384.1990
190.0879
204.1039
192.0974
368.1868
366.1717
384.1815
410.1975
338.1375
354.1733
410.2360
424.2489
354.1710
194.1180
208.1337
208.1337
236.1649
333.2666
332.2588
450.2431
478.2744
478.2744
506.3057
476.2587
176.0711
190.0867
204.1024
232.1337
288.1962
288.1962
316.2275
288.1962
302.2118
370.1805
384.1962
190.0867
204.1023
192.0999
368.1860
366.1703
384.1809
410.1965
338.1391
354.1704
410.2330
424.2485
354.1704
6.8b
13.5b
11.5b
36.4b
23.1c
21.3c
29.7c
32.5c
26.2c
34.0c
23.2c
12.3b
18.9b
29.0b
25.6c
27.2c
14.4c
30.0c
17.3c
18.7c
20.2c
20.7c
13.7b
20.1b
21.9b
10.0d
11.1d
8.4d
3.5b
7.1b
5.9b
26.9b
17.4c
16.5c
19.9c
20.8c
20.1c
22.8c
20.2c
7.2b
e
C14H22NO2
C21H35NO2
f
e
17
95
C21H34NO2
C31H32NO2
C33H36NO2
C33H36NO2
C35H40NO2
C33H34NO2
e
e
10
11
12
13
14
15
16
18
20
21
22
23
24
25
26
27
28
29
42
43
44
45
46
47
48
49
50
85
e
70
38
e
e
MeOH–C6H6
41
59
45f
33
10
58
48
MeOH–(CH3)2CO
MeOH–(CH3)2CO
MeOH–(CH3)2CO
C10H10NO2
C11H12NO2
C12H14NO2
11.8b
19.8b
10.6c
16.8c
16.2c
19.7c
14.6c
16.0c
16.9c
17.7c
9.2b
e
C14H18NO2
C18H26NO2
C18H26NO2
C20H30NO2
C18H26NO2
C19H28NO2
C25H24NO2
C26H26NO2
e
Et2O–(CH3)2CO
MeOH
e
e
40
e
EtOH
Et2O–(CH3)2CO
MeOH–(CH3)2CO
MeOH–(CH3)2CO
(CH3)2CO
54g
29
e
85–87
e
220–229
187–200
254–264
196–211
200–201
149–155
86–100
169–177
193–198
184–190
114–119
205–210
204–212
144–146
88
22h
85
e
C11H12NO2
C12H14NO2
C11H14NO2
C22H26NO4
C22H24NO4
C22H26NO5
C24H28NO5
C20H20NO4
C21H24NO4
C25H32NO4
C26H34NO4
C21H24NO4
(CH3)2CO
78
14.4b
12.7b
8.5d
EtOAc–(CH3)2CO
MeOH–(CH3)2CO
(CH3)2CO
MeOH–(CH3)2CO
MeOH–(CH3)2CO
(CH3)2CO
EtOAc
MeOH–(CH3)2CO
MeOH–(CH3)2CO
85
99
70
93
98
80
76
55
70
9.6d
7.6d
11.0d
11.4d
10.5d
16.9d
17.9d
10.9d
9.5d
9.8d
8.8d
14.8d
15.2d
9.3d
aI: 0.1 M NH4OAc/MeOH (0.05% TFA) A/B, II; H2O/MeOH (0.05%TFA) A/B.
bInitial A/B 95/5, 30 min 80/20.
cInitial A/B 80/20, 30 min 0/100.
dInitial A/B 75/25, 10 min 50/50, 20 min 80/20.
eWhen the compound was purified by preparative HPLC, the anion was CF3COOꢀ.
fFree base.
gYield from 2.
hYield from 15.

K. Iwasa et al. / Bioorg. Med. Chem. 9 (2001) 2871–2884
2879
Table 4. Antibacterial and antifungal activities of 1-alkyl substituted isoquinolines and 1- or 3-benzylisoquinolines
Compd
MIC (mg/mL)a
S. enteritidis
S. aureus
B. subtilis
E. coli (IFO 026)
C. albicans (IFO 1061)
1–5
6
7
8, 10–11
13
>500
>500
250
>500
3.9
>500
>500
250
>500
3.9
000
>500
250
>500
>500
>500
>500
15.6
>500
>500
> 500
> 500
3.9
>500
3.9
14–16, 18
20–22
23
24
25
>500
>500
250
>500
>500
500
>500
>500
250
>500
>500
>500
>500
250
> 500
>500
>500
>500
250
250
250
125
31.3
15.6
>500
>500
>500
125
62.5
31.3
>500
>500
>500
31.3
31.3
3.9
31.3
15.6
>500
>500
>500
125
26
125
250
27, 28
30–39
40–47, 50
48
49
KAb
>500
>500
>500
>500
>500
31.3
>500
>500
>500
>500
>500
>2000
125
31.3
62.5
62.5
aThis value was defined as the lowest concentration of the test substance that did not induce growth in comparison with a blank experiment.
Table 5. In vitro antimalarial activity of 1-alkyl substituted isoquinolines and 1- or 3-benzylisoquinolines
Compd
50% Inhibitory concentration (mol)a
Selectivity indexb IC50/EC50
Plasmodium falciparum
FCR-3 EC50 (growth%)
Mouse mammary cells
FM3A IC50 (growth%)
1
2
3
4
5
7
(81.5)
(69.2)
(76.7)
(100)
(81.5)
>5.2E-5 (96.2)
>4.8E-05 (88.9)
>8.2E-05 (88.2)
<8.7E-05 (81.6)
>3.8E-05 (79.8)
3.5E-06
>4
0.4
1
1.3
0.9
0.7
5
5
>43
29
0.9
0.8
1.2
>2
>1
>32
>3
>1
>1
>2
>2
>3
>17
>3
910
8.2E-06
4.0E-07
1.3E-05
1.7E-06
1.8E-05
4.6E-07
4.6E-07
3.0E-07
1.7E-07
7.7E-06
5.9E-06
8.5E-06
5.8E-06
8.2E-06
3.1E-07
4.0E-06
9.2E-06
8.3E-06
4.10E-06
5.0E-06
3.6E-06
5.8E-07
4.0E-06
1.1E-07
13
14
15
16
23
24
25
26
27
28
30
40
41
42
43
44
45
46
47
48
49
50
Quinine
3.0E-07
1.7E-05
1.6E-05
1.2E-05
2.1E-06
2.4E-06
>1.3E-05 (74)
5.0E-06
7.2E-06
5.0E-06
1.0E-05
>1.0E-05 (100)
>1.0E-05 (100)
>1.0E-05 (100)
>1.0E-05 (96)
>1.0E-05 (100)
>1.0E-05 (100)
>1.0E-05 (100)
>1.0E-05 (100)
>1.0E-05 (100)
>1.0E-05 (100)
>1.0E-05 (100)
> 1.0E-04
aThe 50% inhibitory concentration was defined by comparison with drug-free controls incubated under same conditions.
bIn vitro selectivity index was estimated from the ratio (IC50/EC50) of the drug concentrations necessary to inhibit the growth rate of cells to 50% of
the growth value between the malaria parasites and mouse mammary FM3A cells which served as a model host.

2880
K. Iwasa et al. / Bioorg. Med. Chem. 9 (2001) 2871–2884
Table 7. Anti-HIV activity of simple isoquinolines and 1- or 3-ben-
zylisoquinolines
Table 6. In vitro cytotoxicity of simple isoquinolines and 1- or 3-
benzylisoquinolines against various human tumor Cell linesa
c
Compd
EC50 (mg/mL)a
IC50 (mg/mL)b
TI (IC50/EC50)
Compd
ED50 (mg/mL)b
1
2
3
4
0.117
1.52
2.88
3.21
NSd
NS
NS
NS
NS
4.98
NS
NS
<0.10
<0.10
NS
NS
NS
NS
NS
8.04
82.8
NS
NS
NS
NS
NS
NS
NS
21.2
20.9
21.8
22.8
181
13.7
7.57
7.11
—
—
—
—
—
1.08
—
—
20.7
236
—
—
—
—
—
KB A-549
>4 >4
HCT-8
1A9 MCF-7 U-87 MG
1–
13
14
15
16
23
25
27
>4
>4
0.5
>4
1.8
> 4
4.4
1.1
5.3
9.0
2.7 > 1 (45)
10.0 3.8
8.5 4.2
>4 >4
16.0
>1 (45)
3.4
>4
5
21.6
4.2 10.0 >20 (46)
4.8 10.0 >20 (42)
13e,f
14e,f
15
0.197
2.22
2.08
2.23
5.40
0.171
2.15
2.07
23.6
11.6
1.85
18.8
21.6
10.2
8.48
>4
>4
0.8 10.0
0.1 1.0
8.9 10.0 > 20 (47)
>4
>4
18.5
0.5
3.1
0.4
8.3
6.5
0.3
18.5
16
23
25e
27e
28
28, 29
30–39
40–50
>4
>4
>4
>4
>4
—
>4
>4
>4
>0.4
>4
>4
>4
—
>4
>4
>4
>4
>4
>4
0.016
29
Colchicine 0.002 0.002
31
32e
33
aKB: epidermoid carcinoma of the nasopharynx, A-594: lung carci-
noma; HCT-8: ileocecal carcinoma; 1A9: ovarian cancer; MCF-7:
breast cancer; U-87 MG: glioblastoma.
bCytotoxicity as ED50 for each cell line, the concentration of com-
pound that causes a 50% reduction in adsorbance at 562 nm relative
to untreated cells using the SRB assay.27 Pure compound is considered
to be significantly active when its ED50 < 4.0 mg/mL.Percent inhibi-
tion of compound with less than 50% inhibition at the highest con-
centration tested is given as the bracketed value.
35
36
37e
38e,f
39
1.05
> 1.21
—
—
—
—
—
—
—
—
—
—
38,462
>100g
10.1
42
43
>100g
17.4
44
>100g
20.6
22.4
45e
46
47
>100g
17.1
13.0
The human tumor cells HCT-8 or IA9 appear to be the
most sensitive in detecting active compounds of the
simple isoquinolines among the six cell lines tested in
this screening study.Human KB cells also are fairly
sensitive.
48e
49e
50
NS
NS
NS
>100g
500
AZTh
0.013
aThe agent concentration that inhibited viral replication in H9 cell by
50%.
All tested benzylisoquinolines (30–39) and their N-
methylated quaternary salts (40–50) displayed low
cytotoxicity (ED50 >4.00 mg/mL).
bThe agent concentration that inhibited H9 cell growth by 50%.
cIn vitro therapeutic index (TI) ratio: IC50/EC50.
dNS=no suppression.
eCrystals were seen at 100 mg/mL concentration by light microscopy
for these agents.
fCrystals were also seen at 10 mg/mL concentration by light micro-
scopy for these agents.
Anti-HIV activity
gBecause all agents were dissolved in DMSO and their stock con-
centrations were 10 mg/mL, a minimum of a 1:100 dilution was nee-
ded to negate the effects of DMSO on this assay; thus, the greatest
agent concentration possible was 100 mg/mL.
hAzidothymidine.
Fourteen simple isoquinolines (1–5, 13–16, 23, 25, 27–
29) and 17 benzylisoquinolines (31–33, 35–39, 42–50)
were tested against HIV-1 replication in H9 lympho-
cytes.The data are shown in Table 7.Compounds 1, 28,
and 29 were most potent with ED50 values of 0.117 mg/
mL (1) and <0.10 mg/mL (28, 29).Among the 6,7-
dihydroxy-tetrahydroisoquinolines, anti-HIV activity,
but not cytotoxicity, decreased as the C-1 alkyl chain
lengthened.The potent 1-methyl derivative ( 1) had an
ED50 value of 0.117 mg/mL and an IC50 value of 21.2
mg/mL giving a calculated therapeutic index (TI)
[defined as toxicity (IC50) divided by anti-HIV activity
(EC50)] of 181, while the 1-pentyl derivative (5) was
inactive.The 6,7-dihydroxy isoquinolines ( 14–16) also
were inactive, as was the O,N-tribenzyl dihy-
droisoquinolinium salt 13.Isoquinolinium salts bearing
an ethyl group at C-1 (23 and 28) were active with EC50
values of 4.98 and <0.10 mg/mL, respectively.They
inhibited uninfected H9 cell growth with IC50 values of
5.40 and 2.07 mg/mL, respectively, giving TI values of
1.08 and 20.7, respectively. However, interestingly,
similar isoquinolinium salts having a methyl group at C-
1 (25 and 27) showed no anti-HIV activity.Among all
tested compounds, 28 and 29 were most potent with an
EC50 of < 0.10 mg/mL.The latter compound was most
selective with an IC50 of 23.6 mg/mL and a TI of 236.
These results suggested that the bulkiness of the chain at
C-1 modulated the activity.Interestingly, results in the
anti-HIV assay did not parallel those in the anti-
microbial, antimalarial, and cytotoxicity assays.The
most potent compounds (13, 25) in the latter assays
were inactive against HIV replication.
Among the 17 tertiary and quaternary benzylisoquino-
lines, only tertiary benzylisoquinolines 37 and 38
showed weak anti-HIV activity.
Conclusions
Regarding SARs, some common features were found
among antimicrobial, antimalarial, and antitumor activ-
ities of the simple isoquinolines.A quaternary nitrogen
atom of isoquinolinium or dihydroisoquinolinium type

K. Iwasa et al. / Bioorg. Med. Chem. 9 (2001) 2871–2884
2881
contributed to increased potency in all three types of
activities.Benzylation of the C-6 and C-7 hydroxy
groups also enhanced activity, while propylation
increased activity to a lesser extent.In contrast, anti-
HIV activity was found with tetrahydroisoquinolines
and 6,7-dihydroxy-isoquinolinium salts.
CH3I to give N-methylpapaverine 40.Benzylisoquino-
lines, 31,14 32,15 33,16 34,17 35,18 36,19 37,20 38,21 39,22 and
4123 were prepared according to literature procedures.
General procedure for the preparations of 6,7-dihydroxy-
1-alkyl-1,2,3,4-tetrahydroisoquinolines (2–5). To a solu-
tion (pH 4) of dopamine HCl (1 g) and a few drops of
concentrated HCl in H2O (5 mL) was added an appro-
priate aldehyde (1.2 equivalent weight). The mixture
was allowed to stand at room temperature for 2–5 days
then evaporated to dryness.The crystalline products
except for 5 were recrystallized from MeOH to give 2–4.
Crude 5 was purified by preparative HPLC to give pure
5.For 1H NMR and physical, LSIMS, HRLSIMS, and
HPLC data, see Tables 1 and 3.
With these present studies, we have identified new bio-
logically active compounds of the isoquinoline type.The
compounds to be considered as lead structures for
developing potential chemotherapeutic agents include
13 (antimicrobial), 25, 26, and 42 (antimalarial), 13 and
25 (cytotoxic), and 28 and 29 (anti-HIV).Further
experiments are under development to explore the cel-
lular processes targeted by these compounds and to
better understand the differences between SAR of the
anti-HIV and antimicrobial/antimalarial/cytotoxic
activities.
1,2-Propyl-6,7-dipropyloxy-1,2,3,4-tetrahydroisoquinoline
(6). To a solution of 3 (500 mg) in MeOH (10 mL) and
EtOH (20 mL) was added K2CO3 (1.5 g) and the mix-
ture was stirred at room temperature for 30 min.Propyl
iodide (1 mL) was added to the mixture, and the solu-
tion was refluxed for 17 h.Water (20 mL) was added,
and the reaction mixture was concentrated under
reduced pressure.The aqueous solution was extracted
with CHCl3.The combined organic layers were dried
(Na2SO4) and evaporated to afford the crude product
(650 mg, 81% yield), which was purified by preparative
HPLC to give 6 (550 mg) as oil.For 1H NMR and
Physical, LSIMS, HRLSIMS, and HPLC data, see
Tables 1 and 3.
Experimental
General methods
Melting points were determined on a Yanako Micro-
1
melting Point apparatus and are uncorrected. H NMR
spectra were recorded on a Varian VXR-500 (500 MHz)
using TMS as an internal standard and CD3OD or
CDCl3 as solvent.Mass spectra were determined on a
Hitachi M-4100 instrument.The secondary ion mass
spectra (LSIMS) were measured using glycerol as
matrix.HPLC and preparative HPLC analyses were
performed on a Hitachi M-6200 intelligent pump (1 mL/
min) and Hitachi M-6250 intelligent pump (6 mL/min),
respectively, with a Hitachi L-4000 UV detector (280
nm).HPLC analyses were also performed on a Jasco
HPLC system, which employed an 880-PU intelligent
pump, 875-UV intelligent UV/VIS detector, 807-IT
integrator, 802-SC system controller, and 860-CO col-
umn oven (40 ꢁC).Cosmosil 5C 18-AR reversed-phase
column of analytical (4.6 i.d.ꢂ150 mm) and preparative
(20 i.d.ꢂ250 mm) columns were used for HPLC.Ana-
lyses with a Hitachi HPLC system were made using two
solvent systems, I: (A) 0.1 M NH4OAc (0.05%TFA)/(B)
MeOH (0.05% TFA); II (A) H2O (0.05% TFA)/(B)
MeOH (0.05% TFA), under the following gradient
conditions: A/B, initial (75/25), 10 min (50/50), 20 min
(20/80).Preparative HPLC analyses were performed
using a solvent system II under the following gradient
condition: A/B 50/50 to 0/100, 30 min.HPLC Analysis
with A Jasco HPLC system were carried out using sol-
vent system, I: (A) 0.1M NH4OAc (0.05%TFA)/(B)
MeOH (0.05% TFA); II (A) H2O (0.05% TFA)/(B)
MeOH (0.05% TFA), under the following gradient
conditions: A/B 95/5 to 80/20, 30 min or 80/20 to 0/100,
30 min.Preparative TLC was carried out on silica gel
plates (Merck, 60F-254).All final compounds were
homogeneous by HPLC analysis; elemental analysis
data are found in Table 8 for selected compounds.
1,2-Propyl-6,7-dipropyloxy-3,4-dihydroisoquinolinium salt
(7). Iodine (500 mg) in EtOH (15 mL) was added drop-
wise to a stirred solution of 6 (200 mg) and CH3COOK
(250 mg) of EtOH (20 mL) for 20 min.Water (20 mL)
was added to the reaction mixture and it was con-
centrated and extracted with CHCl3.The organic layer
was washed with 5% sodium bisulphite, dried
(Na2SO4), and evaporated in vacuo.The residue was
purified by preparative HPLC to give 7 (34 mg) as oil.
For 1H NMR and physical LSIMS, HRLSIMS, and
HPLC data, see Tables 1 and 3.
O,N-Tribenzyl-1-alkyl-1,2,3,4-tetrahydroisoquinolines
(8–12). To a solution of 1 (1 g, 5.6 mM) in MeOH (20
Table 8. Elemental analysis of isoquinolines and benzylisoquinolines
Compd
Formula
Calcd
H
Found
H
C
N
C
N
2
3
4
5
C11H16ClNO2
C12H18ClNO2
C16H18ClNO2
C16H22F3NO4
57.37 6.88 6.05 57.52 7.02 6.10
58.84 7.53 5.83 59.13 7.44 5.75
58.91 7.50 5.66 59.13 7.44 5.75
54.65 6.22 4.02 55.01 6.35 4.01
15
16
27
29
43
45
47
48
49
50
C11H12ClNO2ꢃ 1.5H2O 52.18 5.51 5.51 52.28 5.38 5.54
C12H14ClNO2ꢃ 0.5H2O 58.01 5.78 5.71 57.88 6.07 5.63
C11H12ClNO2ꢃ H2O 54.14 5.70 5.69 54.21 5.79 5.75
C11H14ClNO2ꢃ 3H2O 47.00 6.94 4.87 46.89 7.15 4.97
C22H24ClNO4ꢃ 2H2O 60.22 6.32 3.09 60.33 6.44 3.20
C24H28ClNO5ꢃ 2H2O 59.82 6.55 2.81 59.80 6.69 2.91
C21H24ClNO4ꢃ 1.5H2O 60.91 6.05 3.40 60.50 6.04 3.38
C25H32ClNO4ꢃ 2.5H2O 61.44 7.13 2.85 61.78 7.15 2.88
C26H34ClNO4ꢃ H2O 65.65 7.43 3.02 65.60 7.41 2.94
C21H24ClNO4ꢃ 1.5H2O 60.54 6.13 3.37 60.50 6.16 3.36
Salsolinol (1) was prepared previously.Isoquinoline 29
was synthesized in our laboratory.13 Commercially
available papaverine 30 (Sigma) was N-methylated with

2882
K. Iwasa et al. / Bioorg. Med. Chem. 9 (2001) 2871–2884
mL) and EtOH (40 mL) was added K2CO3 (2.2g, 16
mM) and the mixture was stirred at room temperature
for 30 min.To the mixture was added C H5CH2Cl (2.0
heated for 15 h at 100–110 ꢁC in a oil bath.The solvent
was evaporated to dryness and the resulting crystals
were recrystallized from EtOH to give the iodide (755
mg), which was converted to the chloride 23 by treat-
ment with AgCl in MeOH.For 1H NMR and Physical,
LSIMS, HRLSIMS, and HPLC data, see Tables 1 and
3.
6
g, 16 mM) and it was refluxed for 14 h.Water (40 mL)
was added to the reaction mixture and it was con-
centrated under reduced pressure.The aqueous layer
was extracted with CHCl3.The combined organic layer
was dried (Na2SO4) and concentrated to give the crude
product (2.0 g, 97% yield) which was purified by pre-
parative HPLC to give 8 (1.55 g). Compounds 9–12
were prepared from 2–5 by a similar procedure as for
the synthesis of 8.Compound 9 was used in subsequent
reactions without purification.For 1H NMR and Phy-
sical, LSIMS, HRLSIMS, and HPLC data, see Tables 1
and 3.
1-Propyl-6,7-dipropyloxy-N-methylisoquinolinium salt
(24). This was prepared in a similar manner from 20.
1
For H NMR and Physical, LSIMS, HRLSIMS, and
HPLC data, see Tables 1 and 3.
1-Methyl-6,7-dibenzyloxy-N-methylisoquinolinium chlo-
ride (25). A solution of 14 (545 mg), K2CO3 (2g), and
C6H5CH2Cl (1 mL) in MeOH (10 mL) and EtOH (50
mL) was refluxed for 5 h.Water was added to the mix-
ture then it was concentrated.The aqueous solution was
O,N-Tribenzyl-1-propyl-3,4-dihydroisoquinolinium salt
(13). Iodine (2.3 g) in EtOH (20 mL) was added drop-
wise to a stirred solution of 10 (2.3 g) and CH3COOK (1
g) in EtOH (30 mL) for 30 min.Water (50 mL) was
added to the reaction mixture then it was concentrated
and extracted with CHCl3.The organic layer was
washed with 5% sodium bisulphite, dried (Na2SO4),
and evaporated in vacuo.The crystalline product was
recrystallized from MeOH–C6H6 to give 13 (1.117g).
extracted with CHCl3.The CHCl solution was dried
3
(Na2SO4) and evaporated to dryness.The residue was
purified by a preparative TLC (benzene/Et2O 4:1) to
give 1-methyl-6,7-dibenzyloxyisoquinoline (320 mg,
yield 32%), which was dissolved in acetone (10 mL).
CH3I (1 mL) was added to the solution, and the mixture
was allowed to stand at room temperature for 1 h.The
resulting iodide was filtered and recrystallized from
MeOH–acetone to give the pure iodide (358 mg), which
was converted by treatment with AgCl in MeOH to the
chloride 25.For 1H NMR and Physical, LSIMS,
HRLSIMS, and HPLC data, see Tables 1 and 3.
1
For H NMR and Physical, LSIMS, HRLSIMS, and
HPLC data, see Tables 1 and 3.
1-Alkyl-6,7-dihydroxyisoquinolines (14–18). Crude O,N-
tribenzyl derivative 9 (1.84 g) was heated at reflux with
10% palladium on charcoal (500 mg) in tetraline (60
mL) for 30 min.After cooling, the mixture was filtered
through a small pad of Celite, and the residue was
washed with CHCl3–MeOH.The combined filtrate was
concentrated and extracted with 10% HCl several times.
The aqueous layer was washed with Et2O, filtered, and
evaporated to dryness to give the crystalline product,
which was recrystallized from MeOH–acetone to afford
15 (397 mg).Compounds 14, 16–18 were prepared from
8 and 10–12 in a similar manner.Preparative HPLC was
used for purification of 18.For 1H NMR and Physical,
LSIMS, HRLSIMS, and HPLC data, see Tables 1 and
3.
1-Ethyl-6,7-dibenzyloxy-N-methylisoquinolinium chloride
(26). This was prepared in a similar manner from 15.
1
For H NMR and Physical, LSIMS, HRLSIMS, and
HPLC data, see Tables 1 and 3.
1-Methyl-6,7-dihydroxyisoquinolinium chloride (27). The
chloride 25 (246 mg) was dissolved in 20% HCl (10 mL)
and the mixture was refluxed for 5 h.The aqueous
solution was washed with Et2O and evaporated to dry-
ness.The crystalline residue was recrystallized from
acetone to give 27 (94 mg).For 1H NMR and Physical,
LSIMS, HRLSIMS, and HPLC data, see Tables 1 and 3.
1-Alkyl-6,7-dipropyloxyisoquinolines (19–22). To a solu-
tion of 17 (560 mg) in MeOH (10 mL) and EtOH (20
mL) was added K2CO3 (1.5 g) and the mixture was
stirred at room temperature for 30 min.Propyl iodide (1
mL) was added to the mixture then it was refluxed for
17 h.After water (20 mL) was added, the reaction mix-
ture was concentrated under reduced pressure.The
aqueous solution was extracted with CHCl3.The com-
bined organic solution was dried (Na2SO4) and evapo-
rated to afford the crude product, which was purified by
preparative HPLC to give 21 (464 mg) as oil.Com-
pounds 19, 20, and 22 were prepared by a similar pro-
1-Ethyl-6,7-dihydroxyisoquinolinium chloride (28). This
was prepared in similar fashion from 26.For 1H NMR
and Physical, LSIMS, HRLSIMS, and HPLC data, see
Tables 1 and 3.
1- and 3-Benzyl-N-methylisoquinolinium chlorides (42–
49 and 50) [N-Methylations of 1-benzylisoquinolines
(31–38) and 3-benzylisoquinolines (39)]. To a solution of
31 (1 g) in CHCl3 (2 mL) and MeOH (4 mL) was added
CH3I (2 mL).After standing for 3 days at room tem-
perature, the mixture was evaporated to dryness.The
resulting iodide was converted by treatment with AgCl
in MeOH to the chloride, which was recrystallized from
AcOEt–acetone to give 42 (877 mg).Compounds 32–39
were dissolved in MeOH–acetone or MeOH–CHCl3 and
N-methylated by a similar procedure as in the synthesis
of 42 to yield 43–50, respectively.For 1H NMR and
Physical, LSIMS, HRLSIMS, and HPLC data, see
Tables 2 and 3.
cedure.For
HRLSIMS, and HPLC data, see Tables 1 and 3.
1H NMR and Physical, LSIMS,
1-Ethyl-6,7-dipropyloxy-N-methylisoquinolinium chloride
(23). Crude 1-ethyl-6,7-dipropyloxyisoquinoline (19)
prepared from 15 (967 mg) and 5 mL of CH3I in MeOH
(50 mL) were placed in a glass-stoppered bottle and

K. Iwasa et al. / Bioorg. Med. Chem. 9 (2001) 2871–2884
2883
Antimicrobial assay
tests.The 50% inhibitory concentration (ECI 50) is
defined by comparison with drug-free controls incu-
bated under the same conditions.27,28
Antibacterial activity against S. aureus, B. subtilis, and
S. enteritidis, isolated from hospitalized patients, and E.
coli (IFO 026) and antifungal activity against C. albi-
cans (IFO 1061) were determined by means of the
minimum inhibitory concentration (MIC) using the 2-
fold serial broth dilution test in liquid nutrient medium
and 24-well microplates.MIC was defined as the lowest
concentration of the test substance that did not induce
visible growth in comparison with a blank experiment.
The compounds were dissolved in water–1% DMF.
Dilutions with the test medium furnished concentra-
tions from 1.0 to 500 mg/mL.Blanks were prepared in
water–1% DMF only.Kanamycin sulfate was used as
standard.Each well of 24-well plates contained an
appropriate growth medium with a different concentra-
tion of the respective berberine derivatives.The 24-well
plate was incubated at 37 ꢁC for 24 h for bacteria and at
25 ꢁC for 48 h for the fungus.Bacteria tested were pre-
liminarily cultivated in 3% nutrient broth (‘Nissui’,
Japan) at 37 ꢁC, while C. albicans was cultivated in 3%
malt extract powder (‘Oriental’, Japan) at 25 ꢁC.All
experiments were run in duplicate or triplicate.For
measuring the growth of cells, a constant amount of
sample (200 mL) was transferred to a 96-well test plate
from individual wells (1 mL) of a 24-well plate.After
incubation, the microbial growth was examined by
measuring the optical density at 655 nm with a Model
450 Microplate Reader (Bio-Rad).
Mammalian cells. A wild-type mouse FM3A cell line
(subclone F-28-7) was supplied by the Health Sciences
Research Resources Bank (Osaka, Japan).FM3A cells
were maintained in suspension culture at 37 ꢁC in a 5%
CO2 atmosphere in plastic bottles containing ES med-
ium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan)
supplemented with 2% heat-inactivated fetal bovine
serum (Gibco, NY, USA).29,30
Toxicity to mammalian cells. Cell lines grew with a
doubling time of about 12 h.Before being exposed to
drugs, cells were seeded at a 990 mL of density of 5ꢂ104
cells/mL and various concentrations of compounds dis-
pensed in 10 mL of distilled water were added to indivi-
dual wells of a 24-well plate.The plates were incubated
at 37 ꢁC in a 5% CO2 atmosphere for 48 h.Cell num-
bers were measured using a blood cell counter CC-108
(Toa Medical Electric Co., Japan). All data points
represent means of at least two experimental tests.The
50% inhibitory concentration (IC50) is defined by com-
parison with that of drug-free controls incubated under
the same conditions.Cell growth inhibition is the index
of cytotoxicity including cytostatic activity of the test
compounds.
Selective toxicity. The selective toxicity was estimated
from the ratio (IC50/EC50) of the drug concentrations
necessary to inhibit the growth rate of cells to 50% of
the growth value between the malaria parasites and mouse
mammary FM3A cells, which served as a model host.31
In vitro antimalarial screening
Parasites. In all studies described in this report, P. fal-
ciparum strain FCR-3 (ATCC 30932) was used.24,25
Human serum and erythrocytes were obtained from
healthy donors, stored at 4 ꢁC and used within 10–14
days from donation.Parasites were cultured in 10%
heat inactivated A+ human erythrocytes and suspended
at a 5% hematocrit in RPMI 1640 medium (Gibco, NY,
USA) which contained 50 mg of gentamicin per liter
and 10% group A+ human serum and was buffered
with 25 mM N-2-hydroxyethyl- piperazine-N0-2-ethan-
sulfonic acid (HEPES, pH 7.4) and 25 mM
NaHCO3.19,20 Cultures were maintained at 37 ꢁC in a
gas mixture of 5% oxygen–5% carbon dioxide–90%
nitrogen.26
In vitro cytotoxicity assay. Cytotoxicity was evaluated
using standard HTCL assay.The assay was carried out
according to standard SRB assay procedure described
in Rubinstein et al.32 Samples were prescreened first
against KB at, 40, 4, and 0.4 mg/mL for a two day
exposure period.Active compounds that inhibited KB
cell growth by 40% relative to control at 4 mg/mL, were
re-tested in a dose–response study against HTCL panel.
Drug stock solutions were prepared in DMSO, and the
final solvent concentration was 2% DMSO (v/v), a
concentration without effect on cell replication.The
human tumor cell line panel constituted of epiderimoid
carcinoma of the nasopharynx (KB), lung carcinoma
(A-549), ileocecal carcinoma (HCT-8), renal cancer
(CAKI-1), ovarian cancer (1A9), breast cancer (MCF-
7), and glioblastoma (U-87 MG).Cells were cultured at
37 ꢁC in RPMI-1640 with 100 mg/mL kanamycin and
10% (v/v) fetal bovine serum in a humidified atmo-
sphere containing 5% CO2.Initial seeding densities
varied among the cell lines to ensure a final absorbance
reading in control cultures in the range 1–2.5 A562 units.
Drug exposure was for 3 days, and the ED50 value, the
drug concentration that reduced the absorbance by
50%, was interpolated from dose–response data.
Each test was performed in triplicate, and absorbance
Drug testing. The following procedure was used for
routine assay of antimalarial activity.Various con-
centrations of compounds, suspended in 10 mL of dis-
tilled water were added to individual wells of a 24-well
plate.Erythrocytes with 03.% parasitemia were added
to each well in 990 mL of culture medium to give a final
hematocrit of 3%.The plates were incubated at 37 ꢁC
for 72 h under 5% O2–5% CO2–90% N2.Parasite
morphology in drug-treated culture after 72 h was
measured by staining with Giemsa, and the number of
parasitized red blood cells per 10,000 erythrocytes was
counted and growth rates were calculated.All com-
pounds were run in duplicate at each concentration.
Drug-free control cultures were run simultaneously.All
data points represent means of at least two experimental
readings varied no more than 5%.ED
values
50
determined in independent tests varied no more than
30%.

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K. Iwasa et al. / Bioorg. Med. Chem. 9 (2001) 2871–2884
Anti-HIV assay
References and Notes
1.Preininger, V.In The Alkaloids, 29th ed.; Brossi, A., Ed.;
Academic Press: New York, 1986; pp 1–98.
2.Brossi, A.In The Alkaloids, 43th ed.; Cordell, G. A., Ed.;
Academic Press: New York, 1993; pp 119–183.
3. Szantay, C.; Durnyei, G.; Blasko, G. In The Alkaloids, 45th
ed.; Cordell, G. A., Brossi, A., Eds.; Academic Press: New
York, 1994; pp 128–222.
4. Iwasa, K.; Nishiyama, Y.; Ichimaru, M.; Moriyasau, M.;
Kim, H. S.; Wataya, Y.; Yamori, T.; Takashi, T.; Lee, D. U.
Eur. J. Med. Chem. 1999, 34, 1077.
5. Iwasa, K.; Kim, H. S.; Wataya, Y.; Lee, D. U. Eur. J. Med.
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6. Willets, J.; Lambert, D. G.; Lunec, J.; Griffiths, H. R. Eur.
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7.Arai, Y;.Enomoto, K. Yakugaku Zasshi 1968, 64, 97.
8. Abbasoglu, U.; Bener, B.; Gunay, Y.; Temizer, H. Arch.
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9. Tsai, I. L.; Liuo, Y. F.; Lu, S. T. Kaohsiung J. Med. Sci.
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1988, 4, 336.
11. Boyd, M. R.; Hallock, Y. F.; Cardellina, J. H.; Manfredi,
K. P.; Blunt, J. W.; McMahon, J. B.; Buckheit, R. W., Jr.;
Bringmann, G.; Schaffer, M.; Cragg, G. M.; Thomas, D. W.;
Jato, J.G. J. Med. Chem. 1994, 37, 1740.
12. Iwasa, K.; Kamigauchi, M.; Takao, N. Phytochemistry
1991, 30, 2973.
13. Brossi, A.; Dolan, L. A.; Teitel, S. Org. Synth. 1977, 56, 3.
14. Spaeth, E.; Polgar, N.; Ueber Opium-Alkaloide, V. Ber.
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15.Spaeth, E;.Kruta, E. Ber. Dtsch. Chem. Ges. 1929, 62, 1024.
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17.Pohl, L;.Wiegrebe, W. Z. Naturforsch. 1965, 20b, 1032.
18.Mannich, C;.Walther, O. Arch. Pharm. 1927, 265, 1.
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20. Weijlard, J.; Swanyezy, E. F.; Tashjian, E. J. Am. Chem.
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The T cell line, H9, was maintained in continuous cul-
ture with complete medium [RPMI 1640 with 10% fetal
calf serum (FCS) supplemented with l-glutamine] at
5% CO2 and 37 ꢁC.Aliquots of this cell line were used
in experiments only when in log-phase of growth.Test
samples were first dissolved in DMSO.The following
are the final drug concentrations routinely used for
screening: 100, 20, 4, and 0.8 mg/mL.There were crys-
tals present at the 100 mg/mL concentration of 12, 13,
22, 24, 29, 34, 35, 42, and 46.Crystals were also seen at
10 mg/mL concentration of 12, 13, and 35.For active
agents, additional dilutions are prepared for subsequent
testing so that an accurate EC50 value (see definition
below) could be achieved.As the test samples were
being prepared, an aliquot of H9 cells was infected with
HIV-1 (IIIB isolate), while another aliquot was mock-
infected with complete medium.The mock-infected
sample was used for toxicity determinations (IC50, see
definition below).The stock virus used for these studies
typically had a TCID50 value of 104 Infectious Units
(IU)/mL.The appropriate amount of virus for a multi-
plicity of infection between 0.1 and 0.01 IU/cell was
added to the first aliquot of cells.The other aliquot of
cells received only culture medium and was then incu-
bated under identical conditionsꢁ to the HIV-infected
cells.After a 4-h incubation at 37 C and 5% CO2, both
cell populations were washed three times with fresh
medium and then added to the appropriate wells of a
24-well plate containing the various concentrations of
the test drug or culture medium (positive infected con-
trol/negative-control drug).In addition, AZT was also
assayed during each experiment as a positive-control
drug.The plates were incubated at 37 ꢁC and 5% CO2
for 4 days.Cell-free supernatants were collected on day
4 and tested by an in-house p24 antigen ELISA assay;
p24 antigen is a core protein of HIV and, therefore, is
an indirect measure of virus present in the supernatants.
Toxicity was determined by performing cell counts by a
coulter counter on the mock-infected cells, which had
either received culture medium (no toxicity) or test
sample or AZT.If a test sample had suppressive cap-
ability and was not toxic, its effects were reported in the
following terms: IC50, the concentration of test sample
that was toxic to 50% of the mock-infected cells; EC50,
the concentration of the test sample that was able to
suppress HIV replication by 50%; and therapeutic index
(TI), the ratio of IC50 to EC50.
23.Schneider, W;.Schroeter, K. Ber. Dtsch. Chem. Ges. 1920,
53, 1459.
24.Trager, W;.Jensen, J.B.
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26. Winter, R. W.; Cornell, K. A.; Johnson, L. L.; Igna-
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27. Ofulla, A. V. O.; Okoye, V. C. N.; Khan, B.; Githure, J. I.;
Roberts, C. R.; Johnson, A. J.; Martin, S. K. Am. J. Trop.
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Acknowledgements
This work was supported by a Grants-in-Aid for Scien-
tific Research on Priority Areas (08281105) from the
Ministry of Education, Science, Culture, and Sports,
Japan awarded to Y.Wataya and, in part, by grants
from the National Cancer Institute (CA 17625) and
from the National Institute of Allergy and Infectious
Diseases (AI 33066) awarded to K.H.Lee.