Molecular modeling of 3-arylisoquinoline antitumor agents active against A-549. A comparative molecular field analysis study

Article abstract of DOI:10.1016/S0968-0896(02)00137-2

A series of 58 3-arylisoquinoline antitumor agents were investigated for defining the pharmacophore model using comparative molecular field analysis (CoMFA) program. The studied compounds related to bioisostere of benzophenanthridine alkaloid were synthes

Full text of DOI:10.1016/S0968-0896(02)00137-2

Bioorganic & Medicinal Chemistry 10 (2002) 2953–2961
Molecular Modeling of 3-Arylisoquinoline Antitumor Agents
Active Against A-549. A Comparative Molecular
Field Analysis Study
Won-Jea Cho,a,* Eui-Ki Kim,a Il Yeong Park,b Eun Young Jeong,a Tae Sung Kim,a
d
Thanh Nguyen Le,a Dae-Duk Kimc andEung-Seok Lee
aCollege of Pharmacy, Chonnam National University, Yongbong-dong Buk-gu, Kwangju 500-757, Republic of Korea
bCollege of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea
cCollege of Pharmacy, Pusan National University, Pusan 609-735, Republic of Korea
dCollege of Pharmacy, Yeungnam University, Kyungsan 712-749, Republic of Korea
Received15 January 2002; accepted13 April 2002
Abstract—A series of 58 3-arylisoquinoline antitumor agents were investigated for defining the pharmacophore model using com-
parative molecular field analysis (CoMFA) program. The studied compounds related to bioisostere of benzophenanthridine alka-
loidwere synthesizedandevaluatedfor antitumor cytotoxicity against human lung tumor cell (A 549). In order to perform the
systematic molecular modeling study of these compounds, the conformational search was carried out based on the single X-ray
crystallographic structure of 7,8-dimethoxy-3-phenylisoquinolin-(2H)-one (2). Interestingly, two types of structures having different
dihedral angles between the isoquinoline ring and 3-aryl ring were found in the crystals. Therefore, CoMFA was performed two
different, overlapping ways. The alignments of the structures were based on the common isoquinoline ring and 3-aryl ring. The
3-D-quantitative structure–activity relationship study resulted in significant cross-validated, conventional r2 values equal to 0.715
and0.927, respectively. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
group on aromatic rings playedan important role for
exhibiting the activities.3 In order to increase water
solubility of the compound, the amide carbonyl group
was modified to various amines which could be con-
vertedto salt forms because the water solubility of the
drugs should be considered for in vivo or clinical
experiments. As we expected, the modification of carbonyl
amide group to amines gave relatively potent cytotoxi-
cites against several tumor cell lines as well as good
water solubility. Among these compounds, 1-(4-methyl-
piperazinyl)-3-phenylisoquinoline hydrochloride (CWJ-
a-5) was testedin vivo assay using BDF1 mice (P388
New chemotherapeutic agents for a treatment of cancer
from natural compounds have been developed over the
last decade.1 With the finding of potent antitumor
activity of 7,8-dimethoxy-2-methyl-3-(4,5-methylene-
dioxy-2-vinylphenyl)isoquinolin-1(2H)-one (1) which is
a synthetic intermediate for natural benzo[c]phen-
anthridine alkaloid, chelerythrine, we tried to study
structure–activity relationships of 3-arylisoquinolines
against human tumor cell lines.2 Most of the 3-aryliso-
quinoline derivatives exhibited potent cytotoxicities
against five different human tumor cell lines. The sty-
rene (1) is considered to be a bioisostere of benzo[c]-
phenanthridine which has exhibited potent antitumor
activity by inhibiting DNA topoisomerase I or II.2
From earlier studies, we could obtain the results that the
amide carbonyl group of isoquinoline ring and alkyl
3
leukemia) andresultedin 160 T/C% with low toxicity.
Moreover, the high oral bioavailability of CWJ-a-5 indi-
catedthe possibility of it being developedinto an oral
formulation. Also, the pharmacokinetic data served as
useful information in future clinical studies of CWJ-a-5.4
CoMFA, which is a ligand-based drug design skill, is
not only one of the most used3-D-QSAR methods, but
also has been appliedto a number of different classes of
compounds.5
*Corresponding author. Tel.: +82-62-530-2933; fax: +82-62-530-
2911; e-mail: wjcho@chonnam.ac.kr
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00137-2

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W.-J. Cho et al. / Bioorg. Med. Chem. 10 (2002) 2953–2961
Scheme 1. The structure of 3-arylisoquinolines.
In the previous paper, we reporteda comparative
molecular fieldanalysis (CoMFA) result of 29 3-aryliso-
quinolines with human melanoma tumor cell (SK-
MEL-2) to produce a hypothetical pharmacophore
model.6 In the present study, the model for A-549 tumor
cell line is extended to include various substituents of
the aromatic ring as well as C1 position because it is well
recognized that 3-D QSAR study of diverse modified
molecules, which show a wide range of activities, affords
a reliable pharmcophore model. Therefore, new mole-
cules were designed and synthesized based on the pos-
tulated pharmacophore model derived from former
CoMFA study. Moreover, we tried to determine the
X-ray crystallographic structure of 7,8-dimethoxy-
3-phenylisoquinolin-(2H)-one (2) for getting the infor-
mation of the torsion angle of 3-aryl ring, which is con-
sidered to be crucial for performing CoMFA of the
3-arylisoquinoline derivatives (Scheme 1). Five different
CoMFA pharmacophore contour maps obtainedusing
A-549, SK-OV-3, SK-MEL-2, XF 498 andHCT 15
tumor cell lines showedvery similar patterns. Although
the target sites of action of the compounds were not
clarifiedyet, we assumedthat they share the same
binding sites because of their closely related structures.7
from the corresponding amide was treated with various
amines or thioacetic acidin refluxing DMF to provide
the 1-substitutedisoquinolines in goodyield. For the
preparation of primary amines (6f–6h), the benzyl-
amines (6a–6c) was performedto a catalytic hyrdo-
genation reaction with 5% Pd/C in 80 psi hydrogen
atmosphere to affordthe corresponidng amines in
moderate yield. The coupling reaction of 2,3-dimethoxy-
6,N-dimethylbenzamide (8) with benzonitrile (9) was
accomplishedvia dilithio species using n-BuLi in 62%
yield. The cyclization reaction of 3,4-methylenedioxy-
6,N-dimethylbenzamide (10) and2-methylbenzonitrile
(11) was also performedto yieldthe corresponding iso-
quinoline which was then treatedwith MeI to give the
N-methylatedisoquinoline ( 12) in 63% yield.
Biological data
Cytotoxicity (IC50 value) was obtainedby the following
method: All experimental procedures were followed up
using the NCI (USA)’s protocol basedon the sulforho-
damine B (SRB) method.8 Briefly, tumor cells were cul-
turedto maintain logarithmic growth by changing the
medium 24 h before cytotoxicity assays. On the day of
the assay, the cells were harvestedby trysinization,
counted, diluted in media and added to 96-well plates.
The concentration of tumor cells (A-549) usedwas 1 ꢀ
104. The cells were then preincubatedfor 24 h in a 5%
CO2 incubator at 37 ꢁC. The compounds dissolved in
DMSO were added to the wells in six 2-fold dilutions
starting from the highest concentrations, andincubated
for 48 h in a 5% CO2 incubator at 37 ꢁC. The final
Methods
Synthesis
Synthesis of diverse 3-arylisoquinoline analogues is
described in Scheme 2. The imine chloride (5)6 derived

W.-J. Cho et al. / Bioorg. Med. Chem. 10 (2002) 2953–2961
2955
Scheme 2. Synthesis of 3-arylisoquinoline derivatives.
DMSO concentration was 0.05%. At the termination of
the incubation, the culture medium in each well was
removed, and the cells were fixed with cold 10% tri-
chloroacetic acid(TCA) for 1 h at room temperature.
The microplates were washed, dried, and stained with
0.4% SRB in 1% acetic acidfor 30 min at room tem-
perature. The cells were washedagain andthe bound
stain was solubilizedwith 10 mM Tris base solution (pH
10.5), andthe absorbances were measuredspectro-
photometrically at 520 nm on a microtiter plate reader
(Molecular Devices, Sunnyvale, CA, USA). The data
was transformedinto Lotus-123 format andsurvival
fractions were calculatedby regression analysis (plotting
the cell viability versus the concentration of the test
compound). The IC50 values represent the concen-
trations of the compounds that inhibit 50% of cell
growth. All data represents the average values for a
minimum of three wells (Table 1).
7,8-dimethoxy-3-phenylisoquinolin-1(2H)-one (2). Yel-
lowish transparent prisms were obtainedfrom an ethyl-
acetate solution. The crystal was mountedon a
MacScience KAPPA goniometer equippedwith 18KW
rotating anode. Cell constants were determined as fol-
lows: monoclinic crystal system, a=13.804(2),
b=15.683(4), c=12.632(2) A, ꢀ=93.87(2)ꢁ, space group;
P21/a, Z=8. A total of 4403 independent reflections
were measured. The structure was solved by direct
methodandrefinedby full matrix least-squares proce-
dure to the final R-value of 0.054 with maXus1.1 soft-
ware package.9 There are two independent molecules in
an asymmetric unit of the crystal as shown in Fig. 1.
Two types of conformers having different dihedral
angles between isoquinoline and3-aryl rings were
found in crystal. As depicted, there are three torsion
angles on the molecules such as conformer 1 (t1:
ꢂ150.0ꢁ, t2: 95.0ꢁ, t3: ꢂ179.4ꢁ) andconformer 2 ( t1:
174.6ꢁ, t2: 99.1ꢁ, t3: 2.2ꢁ) shown on the right andleft
side, respectively, in Figure 1.
X-ray crystallographic structures and superimposition
In order to assign the torsion angle of 3-arylisoquino-
lines, we carriedout a single crystal X-ray analysis of
As the initial conformations of the compounds, the
X-ray crystallographic data of 2 was utilized. Due to the

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W.-J. Cho et al. / Bioorg. Med. Chem. 10 (2002) 2953–2961
two different conformations of the molecule 2, we tried
to use both of them as leadtemplates for determining
the conformation of the molecules as shown in Figure 2.
The conformations of the compounds seem to be affec-
tedby t1 value of 3-aryl ring rather than others. To
investigate the conformation profile, we carriedout an
extensive conformational search of the compounds
using the torsion angle of conformers 1 and2. First, the
torsion angle of 3-aryl ring (t1) in the compounds was
fixedas the value ( ꢂ150.0ꢁ) of conformer 1 andthe
conformation of each molecule was obtainedafter per-
forming the energy minimization process. For the opti-
mizedcoordinates, atomic charges were calculatedusing
Gasteiger–Huckel. Secondly, by performing the above
energy minimization process using t1 (174.6ꢁ) of con-
former 2, the active conformers of the compounds were
also obtained. Each active conformer of the 58 com-
pounds was superimposed with that of the reference
molecules (conformer 1 andconformer 2 in Fig. 1) so
that each structural component is as close as possible to
the corresponding components of the reference. Thus,
nine atoms, that is A ring andC3–C4 atoms andthe
C14 atom of C ring were selected, and the sum of the
Table 1. Observedandcalculatedcytotoxicity values against A-549
of the 3-arylisoquinoline derivatives
PIC50
No CompdR1
R2
R3
ObCsdalcDdiff
1
2
3
4
5
6
7
8
1a
—
Me
Me
Me
Me
H
—
Et
—
H2
H2
O
O
O
H
H
H
H
8.05 8.15 ꢂ0.10
3aa
3ba
3ca
3da
3ea
5.89 6.20 ꢂ0.31
5.72 5.80 ꢂ0.08
6.49 6.52 ꢂ0.03
Vinyl
CH2CH(OMe)2
Et
7.57 6.90
0.67
Et
4-OMe
4-Cl
2-PMB
3-Me
4-Br
4-Me
4-OMe
H
4.93 5.27 ꢂ0.34
4ab 5-NMe2
4.90 4.53
5.24 4.85
0.37
0.39
4bb
4cb
4db
4eb
4fb
4gb
4hb
4ic
6-Me
H
H
H
H
H
H
H
H
H
H
H
H
9
4.12 4.29 ꢂ0.17
4.39 4.45 ꢂ0.06
4.21 4.43 ꢂ0.22
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
H
H
H
H
4.52 4.42
0.10
3.81 4.32 ꢂ0.51
3.91 3.43
3.87 3.75
0.48
0.12
2-CHO
2-CH2OH
2-CH2OPMB
2-CH2CH(OMe)2
2-vinyl
H
Me
Me
Me
Me
Me
Me
—
—
—
—
—
—
—
—
Bn
Bn
Bn
Bn
4jc
3.84 3.95 ꢂ0.11
4kc
4lc
4.67 4.66
3.69 3.66
4.22 4.15
0.01
0.03
0.07
4mc
4nb
4.07 4.25 ꢂ0.18
5ab 5-NMe2
4-Cl
2-Cl
2-Me
4-Me
H
3-Me
4-Br
H
2-Me
2-Me
H
3-Me
H
2-Me
2-Me
H
3.84 3.89 ꢂ0.05
5bb
5cb
5db
5eb
5fb
5gb
5hb
6ad
6bd
6cd
6dd
6ed
6fd
6gd
6hd
6id
6-Me
6-Me
6-Me
6-Me
H
H
H
6-Me
H
H
6-Me
H
6-Me
H
H
H
3.88 3.85
0.03
3.58 3.86 ꢂ0.28
3.95 4.24 ꢂ0.29
3.80 3.95 ꢂ0.15
3.77 3.75
3.95 3.71
3.82 3.44
4.56 4.39
3.85 3.78
0.02
0.24
0.38
0.17
0.07
3.76 3.82 ꢂ0.06
6.13 6.28 ꢂ0.15
morpholine 3.91 3.96 ꢂ0.05
NH2
NH2
NH2
6.76 6.25
0.51
5.50 5.70 ꢂ0.20
5.56 5.80 ꢂ0.24
H
H
NH-PMB 3.61 3.59
piperidine 3.83 4.25 ꢂ0.42
0.02
6jd
H
7ab 5-NMe2
7bb 5-NMe2
7cb 5-NMe2
7db 5-NMe2
7eb 5-NMe2
7fb 5-NMe2
2-Me
3-Me
4-Br
4-Cl
4-OMe
H
2-Me
4-Cl
4-Me
H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4.67 4.86 ꢂ0.19
5.03 4.91 0.12
5.04 5.17 ꢂ0.13
5.13 5.13
Figure 1. The crystal structure of 7,8-dimethoxy-3-phenylisoquinolin-
1(2H)-one (2) drawn by ORTEP.10 The thermal ellipsoids are drawn at
the 50% probability level. Hydrogen atoms are drawn as small circles
of arbitrary radii. Two independent molecules in the asymmetric unit
are designated as Conformer 1 and 2, respectively.
0
4.72 4.77 ꢂ0.05
4.83 4.62
5.03 4.86
5.41 5.29
0.21
0.17
0.12
7gb
7hb
7ib
6-Me
6-Me
6-Me
6-Me
H
H
H
H
H
H
H
—
—
—
4.95 5.29 ꢂ0.34
7jb
5.13 5.09 0.04
4.23 4.30 ꢂ0.07
4.67 4.94 ꢂ0.27
7kb
7lb
2-Me
3-Me
4-Br
7mb
7nb
7ob
7pb
7qb
12d
13d
14d
4.73 4.65
4.68 4.59
4.90 4.68
4.75 4.53
4.47 4.38
4.63 4.52
0.08
0.09
0.22
0.22
0.09
0.11
4-Cl
4-Me
4-OMe
H
—
—
3.69 3.70 ꢂ0.01
—
4.60 4.77 ꢂ0.17
aFrom ref 2.
bFrom ref 6.
cFrom ref 13.
Figure 2. Torsion angles of 7,8-dimethoxy-3-phenylisoquinolin-1(2H)-
one (2).
dNewly synthesizedcompounds.

W.-J. Cho et al. / Bioorg. Med. Chem. 10 (2002) 2953–2961
2957
latent variables (components) to be usedin conven-
tional analyses was chosen on the basis of the highest
cross-validated r2 (rc2v) value, the smallest standard error
of prediction (SEP), and the minimum number of com-
ponents. All leave-one-out calculations were carriedout
selecting a 2.0 kcal/mol energy column filter in order to
enhance the signal-to-noise ratio. The steric andelec-
trostatic fieldcolumns were obtainedaccording to the
COMFA-STD default scaling option. In this method, a
fieldis consideredas a whole andevery CoMFA vari-
able is affectedby the overall fieldmean andstandard
deviation. Final PLS (non-cross-validated models) cali-
bration equations were then derived using the optimal
number of components so identified.
Figure 3. Template molecule for molecular superimposition.
squares of the distance of the nine atomic positions
from the corresponding atomic positions of the refer-
ence compoundwas made as small as possible, as shown
in Figure 3.
PKa values of amines
To validate whether or not amines are protonated under
the physiological condition, we calculated their pKa
values using the empirical correlation eqs 1 and2 for
Computer modeling
All computations were done with the molecular model-
ing software package Sybyl 6.6 version.11 The coordi-
nates of the substructures to be modified in substitued
derivatives were calculated with the Sybyl standard
values for bondlengths andangles using the building
module. For the conventional CoMFA, the region was
aliphatic amines (NR1R2R3) reportedby Takayama et
12
al. andHall, respectively.
Eq. 1 is for the primary
andsecondary amines, while eq 2 is for the tertiary
amines.
first createdautomatically with the dimension of 18
ꢀ
Â
Ã
pKa ¼ À3:606 Æ sà þ 0:335 EscðR2Þ þ EscðR3Þ
18 ꢀ 18 A (X=ꢂ7 to 11, Y=ꢂ12 to 6, Z=ꢂ6 to 12).
When the region was manually defined for the mole-
cules to be embedded in the grid box with dimension of
24 ꢀ 22 ꢀ 32 A (X=ꢂ10 to 14, Y=ꢂ13 to 9, Z=ꢂ18
to 14), more reliable statistical values such as r2cv and
SEP were obtainedas shown in Table 2. From this
result, it is suggestedthat the gridsize shouldbe defined
considering that all of the molecules were adequately
fittedin the lattice box. The steric andelectrostatic
potential energies at each lattice point were calculated
using the Coulombic andLennar–dJones potential
function, respectively. The lattice spacing was 2.0 A and
þ 1:427 nH þ 9:545
ð1Þ
ð2Þ
pKa ¼ À3:30 Æ sà þ 9:61
In these equations, Æ s* is the summation of the Taft
values of three N-substituents. Ecs(R2)+Esc(R3) are,
respectively, the Hancock correctedsteric constants of
3
the +1 charge andthe sp carbon were usedas probes
two bulkier substituents R2 andR
among three
3
to estimate the electronic andsteric fields, respectively
with an energetic cutoff of 30 kcal/mol. Regression ana-
lyses were performedusing the Sybyl implementation of
the PLS algorithm, initially with cross-validation (the
leave-one-out technique) to reduce the probability of
obtaining chance correlation. The number of groups of
cross-validation was set as equal to the number of com-
ponents of the training set. The optimal number of
N-substituents, in which R1=H and Ecs(R2)ꢃEsc(R3),
andnH is the number of hydrogen atoms in respective
conjugate ammonium ions. s* and E cs values usedin
the calculations were taken from the literature.12 The
calculation with eqs (1) and(2) estimatedthe p Ka value
as follows: 7a–7q, 10.23; 6j, 11.34. Thus, all the amines
containing the N-methylpiperazinyl group, or primary
amine, are reasonably considered to exist mostly in their
protonated forms under the physiological pH condi-
tions. Therefore, molecular modeling study of amines
was done in their protonated forms.
Table 2. Statistical comparison of the region defined method and
Auto CoMFA with conformer 1 superposition
Alignment Ia
Alignment IIb
CoMFA results
Principal components
r2
SEP
r2
SEP
The CoMFA statistical results for the cytotoxic activ-
ities on A-549 are summarizedin Table 3. In these
equations, m indicates the number of latent variables
and SEP is the standard deviation obtained from the
leave-one-out cross-validation. RC refers to the relative
contribution of steric andelectrostatic effects to varia-
tions in antitumor activity. To determine whether the
hydrophobic parameter is significantly involved, cLogP
values were insertedin the CoMFA analyses. However,
their contribution for the equation was less than 2% in
cv
cv
1
2
3
4
5
6
7
0.023
0.331
0.537
0.624
0.697
0.715
0.685
0.855
0.714
0.600
0.546
0.495
0.485
0.491
0.001
0.281
0.517
0.540
0.616
0.632
0.639
0.865
0.741
0.613
0.604
0.558
0.552
0.552
aRegion defined method; X, ꢂ7 to 11; Y, ꢂ12 to 6; Z, ꢂ6 to 12.
bAuto CoMFA method; X, ꢂ10 to 14; Y, ꢂ13 to 9; Z, ꢂ18 to 14.

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W.-J. Cho et al. / Bioorg. Med. Chem. 10 (2002) 2953–2961
Table 3. CoMFA correlation statistics for 3-arylisoquinolines (n=58)
Type
ma
Nb
Sc
F
r2
Cross-validd
RCe
SEP
q2
Stericf
Elecg
Eq no.
Conformer 1 Superposition/ defined region
Conformer 2 Superposition/ defined region
Conformer 1 Superposition/ auto CoMFA
6
6
7
59
59
59
0.251
0.237
0.251
96.804
109.752
96.804
0.924
0.932
0.924
0.485
0.652
0.552
0.715
0.486
0.639
40.1
46.2
43.2
59.9
53.8
56.8
(3)
(4)
(5)
aNumber of components.
bNumber of compounds.
cStandard deviation.
dObtainedfrom the leave-one-out cross-validation.
eRelative contribution.
fSteric fielddescriptors.
gElectrostatic fielddescriptors.
most cases. So, we initially analyzedfor 58 compounds
andobtainedeqs 3–5. Among these equations, eq 3,
which was derived from the overlapping of the com-
pounds on X-ray conformer 1 as for a lead template,
was the most significant. On the other hand, eq 4 was
obtainedfrom X-ray conformer 2 which gave poorer
statistical results. Therefore, we assumedthat con-
former 1 is the active form for showing the cytotoxicity
andthe molecules shouldhave a ꢂ150ꢁ torsion angle of
atoms of the N-methylpiperazinyl group or primary
amines, whereas a negative electrostatic region, favor-
able to the activity, is locatedat the oxygen atoms of the
carbonyl group and7,8-dimethoxy group. A sterically
forbidden region exists at the C-5 position which
includes the dimethylamino group and N-methylpiper-
azinyl group at C-1. The compounds which have sub-
stituents on C-5 position are less active because their
residues are extended away from the favored region of
interaction. On the other hand, a sterically permissible
region appears at the C-6 position. It is well explained
that methyl substitution at the C-6 position contributes
to the increase of activity.
3-aryl ring. Therefore, the observedpIC
values in
50
Table 1 were calculatedfrom eq 3. Figure 4 represents
the major steric andelectrostatic potential contour
maps drawn according to eq 3 after CoMFA. The green
zone is more important andlocatedaroundthe benzene
ring of the isoquinoline skeleton according to the higher
activity of the molecules. That area indicates regions
where submolecular bulk is well accommodated with an
increase in cytotoxicity, whereas the yellow areas indi-
cate regions where the submolecular bulk is unfavour-
able for activity. The redarea indicates regions where
the more negative electrostatic interaction with the
receptor increases the activity, whereas the blue areas
show regions where the reverse is the case.
Discussion
The application of the comparative molecular field
analysis to a structurally variedset of 58 antitumor
agents resultedin a significant 3-D QSAR model. For
getting higher statistical data, a grid box was constructed
without using the Auto CoMFA method. This work
extends our previous analysis performed on a congeneric
set of isoquinoline analogues andconstitutes a further
step towards a comprehensive 3-D QSAR model of the
cytotoxic activities against A 549 tumor cell line.
In a contour map, a positive electrostatic-potential
region, favoring to activity, appears aroundthe nitrogen
Figure 4. Steric andelectrostatic contour map from the CoMFA model for antitumor 3-arylisoquinolines. Favoring activity: green, bulky group;
yellow, less bulky group; blue, positive charge; red, negative charge.

W.-J. Cho et al. / Bioorg. Med. Chem. 10 (2002) 2953–2961
2959
Figure 5. Plot of the calculatedversus observedpIC
for the CoMFA analysis of the 58 compounds aligned according to eq 3.
50
The descriptive characteristics of the present model are
goodandthe plot of the calculatedversus the observed
pIC50 for the CoMFA of the 58 compounds is shown in
Figure 5.
tography was performedon Merck silica gel 60 (70–
230 mesh). TLC was carriedout using plates coated
with silicagel 60F 254 purchasedfrom Merck Co.
Reagents were obtainedfrom commercial suppliers and
were usedwithout purification.
As regards the steric contours, they show a main region
of positive (green) contribution to the activity that sur-
rounds the 8-methyl group area and this is in agreement
with the cytotoxic activities of the compounds. The
most active compounds such as 1, 3d and 6f couldalso
indicate favorable steric interactions of these structural
elements with a large hydrophobic pocket in the active
site. The 7,8-dimethoxy group and the amide carbonyl
group at the C-1 position were indicated as suitable for
electronegative substitution. The reliability of a QSAR
model is usually evaluated by testing its ability to pre-
dict the biological activity of newly designed molecules
acting on the same biological system. From this study
the CoMFA model not only exhibited that the cyto-
toxicities of the 58 isoquinoline derivatives had an
excellent correlation with the electrostatic andsteric
field, but also provided us with an important pharma-
cophore model which could be useful to consider the
receptor sites three dimensionally.
Benzyl(6-methyl-3-o-tolylisoquinolin-1-yl)amine (6a)
A mixture of 1-chloro-6-methyl-3-o-tolylisoquinoline6
(460 mg, 1.72 mmol), benzylamine (402 mg, 3.75 mmol),
andpotassium carbonate (400 mg, 2.85 mmol) in DMF
was refluxedat 6 h. The reaction mixture was cooledto
room temperature, diluted with water, and extracted
with CH2Cl2. The combinedorganic extracts were
washed with water, dried, and concentrated. The residue
was purifiedby column chromatography on silica gel
with CH2Cl2/MeOH (200:1 to 25:1) to give 6a (420 mg,
72%) as a yellow solid; mp 99–101 ꢁC; IR KBr cmꢂ1
1
3400; H NMR (CDCl3) d 8.00–7.01 (13H, m, Ar–H),
5.40 (1H, s, NH), 4.96 (2H, d, J=5.2 Hz, –CH2–), 2.48,
2.43 (each 3H, each s, Me ꢀ 2); MS, m/e (%) 338 (M+,
39), 340 (27), 336 (23). Anal. C24H22N2 (C, H, N) calcd:
85.17, 6.55, 8.28; found: 85.06, 6.33, 8.56.
Benzyl(6-methyl-3-phenylisoquinolin-1-yl)amine (6b). The
same procedure as described in the preparation of 6a to
give 6b (75%) as a yellow solid; mp 68–69 ꢁC; IR KBr
cmꢂ1 3370; 1H NMR (CDCl3) d 8.18–6.91 (14H, m,
aromatic–H), 5.33 (1H, s, NH), 4.86 (2H, d, J=4.5 Hz,
–CH2–), 2.36 (3H, s, Me); MS, m/e (%) 324 (M+, 39),
321 (19). Anal. C23H20N2 (C, H, N) calcd: 85.15, 6.21,
8.63; found: 85.43, 6.03, 8.78.
Experimental
Melting points were determined on an Electrothermal
IA9200 melting point apparatus andare uncorrected.
Nuclear magnetic resonance spectra (1H NMR) were
recorded on Varian 300 spectrometers, using TMS as
the internal standard ; chemical shifts are reported in
parts per million (d) andsignals are quotedas s (singlet),
d(doublet), t (triplet), q (quartet), andm (multiplet). IR
spectra were recorded on a Perkin-Elmer 783 spectro-
meter anda Nicolet instrument using KBr pellets. Ele-
mental analyses were performedon a CaHo Erba
elemental analyser. Anhydrous tetrahydrofuran (THF)
was distilled from sodium-benzophenon ketyl. Solvents
were routinely distilled prior to use. Column chroma-
Benzyl(3-phenylisoquinolin-1-yl)amine (6c). The same
procedure as described in the preparation of 6a to give
6c (68%) as a yellow solid; mp 122–128 ꢁC; IR KBr
1
cmꢂ1 3400, 1520; H NMR (CDCl3) d 8.20–7.21 (15H,
m, aromatic–H), 5.43 (1H, s, NH), 4.96 (2H, d,
J=5.0 Hz, –CH2–); MS, m/e (%) 310 (M+, 100), 301
(46). Anal. C22H18N2 (C, H, N) calcd: 85.13, 5.85, 9.03;
found: 85.42, 5.63, 9.28.

2960
W.-J. Cho et al. / Bioorg. Med. Chem. 10 (2002) 2953–2961
Benzyl(6-methyl-3-m-tolylisoquinolin-1-yl)amine (6d). The
same procedure as described in the preparation of 6a to
give 6d (65%) as a yellow solid; mp 67–68 ꢁC; IR KBr
cmꢂ1 3340; 1H NMR (CDCl3) d 8.00–7.01 (13H, m,
aromatic–H), 5.45 (1H, s, NH), 4.90 (2H, d, J=5.0 Hz,
–CH2–), 2.54, 2.45 (each 3H, each s, Me ꢀ 2); MS, m/e
(%) 338 (M+, 100), 305 (76), 258 (54). Anal. C24H22N2 (C,
H, N) calcd: 85.17, 6.55, 8.28; found: 85.42, 6.63, 8.55.
6a to give 6i (62%) as a yellow solid; mp 130–131 ꢁC; IR
1
KBr cmꢂ1 3300, 1640, 1476; H NMR (CDCl3) d 8.20–
6.81 (13H, m, aromatic–H), 5.30 (1H, s, NH), 4.87 (2H,
d, J=5.0 Hz, –CH2–), 3.79 (3H, s, OMe); MS, m/e (%)
340 (M+, 100), 339 (32). Anal. C23H20N2O (C, H, N)
calcd: 81.15, 5.92, 8.23; found: 81.35, 5.88, 8.46.
3-Phenyl-1-piperidin-1-yl-isoquinoline (6j). The same
procedure as described in the preparation of 6a to give
6j (88%) as a yellow solid; mp 100–101 ꢁC; IR KBr
1-Morpholin-4-yl-3-phenylisoquinoline (6e). The same
procedure as described in the preparation of 6a to give
6e (95%) as a yellow solid; mp 113–114 ꢁC; IR KBr
1
cmꢂ1 3250, 1650; H NMR (CDCl3) d 8.20–7.34 (10H,
m, aromatic–H), 3.50 (4H, t, J=4.0 Hz, CH2), 1.87 (4H,
t, J=5.6 Hz, CH2), 1.74 (2H, q, J=4.0 Hz, CH2); MS,
m/e (%) 288 (M+, 100), 287 (87), 259 (84). Anal.
C20H20N2 (C, H, N) calcd: 83.30, 6.99, 9.71; found:
83.45, 6.78, 9.66. The solidwas dissolvedin acetone and
c-HCl was added to this mixture to afford the pre-
cipitate which was collectedanddriedin vacuo to give
the hydrochloric acid salt; mp 198–201 ꢁC.
1
cmꢂ1 3150, 1650, 1475; H NMR (CDCl3) d 8.20–7.39
(9H, m, aromatic–H), 7.23 (1H, s, C4–H), 4.01 (4H, t,
J=4.5 Hz, –CH2–), 3.54 (4H, t, J=4.8 Hz, –CH2–); MS,
m/e (%) 290 (M+, 100), 289 (87), 259 (54). Anal.
C19H18N2O (C, H, N) calcd: 78.59, 6.25, 9.65; found:
78.38, 6.46, 9.58.
6-Methyl-3-o-tolylisoquinolin-1-ylamine (6f). The reac-
tion mixture of the benzylamine (6a, 1.14 g, 3.37 mmol)
and5% Pd/C (300 mg) in acetic acid(15 mL) was sha-
ken at 66 ꢁC under H2 (90 psi) using Parr apparatus for
3 days. The reaction mixture was cooled down to room
7,8-Dimethoxy-3-phenyl-2H-isoquinolin-1-one (2). To a
solution of 2,3-dimethoxy-6,N-dimethylbenzamide
(10.25 g, 36.5 mmol) in THF (250 mL) was added
n-BuLi (2.5 M in hexane, 43 mL, 107.5 mmol) at ꢂ20 ꢁC
maintaining the reaction mixture not exceeding 0 ꢁC
under nitrogen. After adding the n-BuLi, the mixture
was stirredfor 1 h at the same temperature. Then, the
color of reaction mixture was turnedto red-purple. The
cooling bath was removedandthe mixture was moved
to ꢂ50 ꢁC cooling bath. Benzonitrile (6.13 g, 59.5 mmol)
in THF (20 mL) was added to the reaction mixture and
then the cooling bath was removedandthe mixture was
stirredovernight at room temperature. The reaction
mixture was quenchedwith excess water at room tem-
perature andthe organic layer was separated, washed
with water and brine, dried, and concentrated to dry-
ness to give the crude isoquinoline as a yellow solid. The
residue was recrystallized from ethyl acetate to give 2 as
a pale yellow needles (6.36 g, 62%); mp 226.4–228.5 ꢁC;
temperature andneutralizedwith satdNaHCO
solu-
3
tion. The extration was done with ethyl acetate and the
combinedorganic layer was washedwith water, brine
anddriedover sodium sulfate. The solvent was evapo-
ratedoff to give the residue which was purifiedby col-
umn chromatography on silica gel with CH2Cl2/MeOH
(100:3) to affordthe free amine as an oil (600 mg, 72%).
The free amine was dissolved in acetone and was added
several drops of c-HCl to affordthe hydrochloric acid
salt of the amine. The resulting precipitate was collected
anddriedin vacuo; mp 269.1–270.2 ꢁC; IR KBr cmꢂ1
3300, 1650; 1H NMR (DMSO-d6) d 13.84, 9.40 (1H and
2H, each s, NH3), 8.79–7.57 (7H, m, aromatic–H), 7.38
(1H, s, C4–H), 2.75, 2.61 (each 3H, each s, Me ꢀ 2); 13
C
NMR (DMSO-d6) d 153.4, 144.4, 137.0, 135.9, 134.8,
131.6, 129.4, 128.7, 128.5, 128.4, 125.7, 124.8, 124.0,
113.0, 109.5; MS, m/e (%) 248 (M+, 100), 232 (87), 230
(54). Anal. C17H17ClN2 (C, H, N) calcd: 71.70, 6.02,
9.84; found: 71.58, 6.27, 9.58.
1
IR KBr cmꢂ1 1651; H NMR (CDCl3) d 11.07 (1H, s,
NH), 7.76–7.42 (7H, m, Ar–H), 6.79 (1H, s, C4–H),
3.86, 3.77 (each 3H, each s, OMe ꢀ 2); MS, m/e (%) 281
(M+, 100), 264 (32). Anal. C17H15NO3 (C, H, N) calcd:
72.58, 5.37, 4.98; found: 72.74, 5.42, 4.83.
3-o-Tolylisoquinolin-1-ylamine (6g). The same procedure
as described in the preparation of 6f to give 6g (63%) as
a yellow oil; mp 232–234 ꢁC (HCl salt); IR CHCl3 cmꢂ1
6-Methyl-7-o-tolyl-6H-[1,3]dioxolo[4,5-g]isoquinolin-5-
one (12). To a solution of 6-methylbenzo[1,3]dioxole-
5-carboxylic acidmethylamied (5.25 g, 27.2 mmol) in
THF (100 mL) was added n-BuLi (2.5 M in hexane,
22 mL, 54.5 mmol) at ꢂ20 ꢁC maintaining the reaction
mixture not exceeding 2 ꢁC under nitrogen. After adding
the n-BuLi, the mixture was stirredfor 4 h at the same
1
3350, 1650, 1475; H NMR (CDCl3) d 7.90–6.85 (9H,
m, aromatic–H), 8.93 (2H, s, NH2), 2.42 (3H, s, Me);
MS, m/e (%) 234 (M+, 100), 232 (87), 215 (54). Anal.
C16H15ClN2 (C, H, N) calcd: 70.98, 5.58, 10.35; found:
70.88, 5.56, 10.56.
temperature. The mixture was cooledto
ꢂ50 ꢁC.
3-Phenylisoquinolin-1-ylamine (6h). The same procedure
as described in the preparation of 6f to give 6h (62%) as
a yellow oil; mp 242–245 ꢁC (HCl salt); IR CHCl3 cmꢂ1
2-Methylbenzonitrile (3.05 g, 29.5 mmol) in THF
(10 mL) was added to the reaction mixture and then the
cooling bath was removedandthe mixture was stirred
overnight at room temperature. The reaction mixture
was quenchedwith excess water at room temperature
andthe organic layer was separated, washedwith water
and brine, dried, and concentrated to dryness to give the
crude isoquinoline as a yellow solid. The residue was
dissolved in THF (20 mL) and NaH (60% dispersion in
oil, 8.7 g, 35 mmol) was added at 0 ꢁC with MeI (4.98 g,
1
3500; H NMR (CDCl3) d 7.90–6.85 (9H, m, aromatic–
H), 8.92 (2H, s, NH2), 2.40 (3H, s, Me); MS, m/e (%) 220
(M+, 100), 210 (87), 168 (54). Anal. C15H13ClN2 (C, H,
N) calcd: 70.18, 5.10, 10.91; found: 70.34, 5.09, 10.66.
(4-Methoxybenzyl)-(3-phenylisoquinolin-1-yl)amine (6i).
The same procedure as described in the preparation of

W.-J. Cho et al. / Bioorg. Med. Chem. 10 (2002) 2953–2961
2961
35 mmol) under nitrogen. The mixture was stirred for
References and Notes
4 h at the same temperature. The reaction mixture was
quenchedwith water andextractedwith ethyl acetate
the combinedorganic layer was washedwith water,
brine and dried over sodium sulfate. The solvent was
evaporatedoff to affordthe residue which was purified
by column chromatography on silica gel with CH2Cl2/
1. Mackay, S. P.; Meth-Cohn, O.; Waich, R. D. In Advances
in Heterocyclic Chemistry, Katritzky A. R., Ed.; Academic:
1997; Vol. 67, p 345.
2. (a) Cho, W. J.; Yoo, S. J.; Chung, B. H.; Cheon, S. H.;
Whang, S. H.; Kim, S. K.; Kang, B. H.; Lee, C. O. Arch. Pharm.
Res. 1996, 19, 321. (b) Cho, W. J.; Yoo, S. J.; Park, M. J.;
Chung, B. H.; Lee, C. O. Arch. Pharm. Res. 1997, 20, 264.
3. Cho, W. J.; Park, M. J.; Chung, B. H.; Lee, C. O. Bioorg.
Med. Chem. Lett. 1998, 8, 41.
4. Kim, K. E.; Cho, W. J.; Chang, S. J.; Yong, C. S.; Lee,
C. H.; Kim, D. D. Int. J. Pharmaceutics 2001, 217, 101.
5. (a) Cramer, R. D. III; Patterson, D. E.; Bunce, J. D. J. Am.
Chem. Soc. 1988, 110, 5959. (b) Raghavan, K.; Buolamwini,
J. K.; Fesen, M. R.; Pommier, Y.; Kohn, K. W.; Weinstein,
J. N. J. Med. Chem. 1995, 38, 890.
MeOH (100:2) to affordthe edsiredcompound
12
(5.82 g, 73%) as a pale yellow solid; mp 148.5–150 ꢁC;
IR KBr cmꢂ1 1650; 1H NMR (CDCl3) d 7.81, 6.81 (each
1H, each s, Ar–H), 7.51–7.20 (4H, m, Ar–H), 6.27 (1H,
s, C4–H), 6.01 (2H, s, –OCH2O–), 3.27 (3H, s, Me), 2.18
(3H, s, –NMe).; MS, m/e (%) 293 (M+, 70), 268 (100).
Anal. C18H15NO3 (C, H, N) calcd: 73.71, 5.15, 4.78;
found: 73.45, 5.38, 4.75.
6. Cho, W. J.; Kim, E. K.; Park, M. J.; Choi, S. U.; Lee, C. O.;
Cheon, S. H.; Choi, B. G.; Chung, B. H. Bioorg. Med. Chem.
1998, 6, 2449.
7. During the preparation of this manuscript, we foundthese
compounds show DNA topoisomerase I inhibitory activities.
Detailedinvestigation relatedto the active sites of the enzyme
is now in progress.
8. (a) Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.;
Simon, R. M.; Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks,
A.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113. (b) Ske-
han, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd,
M. R. J. Natl. Cancer Inst. 1990, 82, 1107.
9. Details of structural results were deposited to the Cam-
bridge Crystallographic Data Center as deposit No. of CCDC
177289. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44
1223 336033; e-mail: deposit@ccdc.cam.ac.uk).
10. Johnson, C. K. ORTEPII: A FORTRAN Thermal-Ellipsoid
Plot Program for Crystal Structure Illustrations (ORNL-5138);
Oak Ridge National Laboratory: Tennessee, USA, 1976.
11. The Sybyl program (Version 6.6) was suppliedby Tripos
Associates, 1699 South Hanley Road, Suite 303, St. Louis, MI
63144, USA.
12. (a) Takayama, C.; Fujita, T.; Nakajima, M. J. Org. Chem.
1979, 44, 2871. (b) Hall, H. K., Jr. J. Am. Chem. Soc. 1957, 79,
5441.
(3-Phenylisoquinolin-1-ylsulfanyl)acetic acid ethyl ester
(13). The same procedure as described in the prepara-
tion of 6a to give 13 (85%) as a yellow solid; mp 69–
70 ꢁC; IR KBr cmꢂ1 1730, 1640; H NMR (CDCl3) d
1
8.14–7.37 (10H, m, aromatic–H), 4.18 (2H, s, –SCH2–),
4.17 (2H, q, J=7.2 Hz, –OCH2CH3), 1.23 (3H, t,
J=7.2 Hz, –OCH2CH3); MS, m/e (%) 323 (M+, 100),
257 (67), 249 (50). Anal. C19H17NO2S (C, H, N) calcd:
70.56, 5.30, 4.33; found: 70.35, 5.38, 4.47.
N,N,N 0 -Trimethyl-N 0 -(3-phenylisoquinolin-1-yl)ethane-
1,2-diamine (14). The same procedure as described in
the preparation of 6a to give 14 (86%) as a yellow solid;
mp 75–77 ꢁC; IR KBr cmꢂ1 1690, 1350; 1H NMR
(CDCl3) d 8.21–7.26 (10H, m, aromatic–H), 3.86 (2H, t,
J=6.5 Hz, –NCH2CH2–), 3.21 (3H, s, NMe), 3.00 (2H,
t, J=6.8 Hz, –CH2CH2ꢂ), 2.51 (6H, s, NMe2); MS, m/e
(%) 305 (M+, 45), 256 (69), 249 (100). Anal. C20H23N3
(C, H, N) calcd: 78.65, 7.59, 13.76; found: 78.45, 7.38,
13.66.
Acknowledgements
This work was supportedby the research grant from the
Ministry of Health andWelfare in Korea (HMP-00-
CH-15–0014).
13. Cho, W. J.; Kim, I. J.; Park, S. J. Bull. Korean Chem. Soc.
2000, 21, 1035.