Inhibition of tubulin polymerization by 5,6-dihydroindolo[2,1- a]isoquinoline derivatives

Article abstract of DOI:10.1021/jm970177c

6-Alkyl-12-formyl-5,6-dihydroindolo[2,1-α]isoquinolines have been shown to inhibit the growth of human mammary carcinoma cells by an unknown mode of action. One of the possible molecular targets is the tubulin system which is involved in Cell division. A

Full text of DOI:10.1021/jm970177c

3524
J . Med. Chem. 1997, 40, 3524-3533
In h ibition of Tu bu lin P olym er iza tion by 5,6-Dih yd r oin d olo[2,1-a ]isoqu in olin e
Der iva tives
Michael Goldbrunner,† Gu¨nther Loidl,† Thomas Polossek,† Albrecht Mannschreck,‡ and Erwin von Angerer*,†
Institut fu¨r Pharmazie and Institut fu¨r Organische Chemie, Universita¨t Regensburg, D-93040 Regensburg, Germany
Received March 17, 1997X
6-Alkyl-12-formyl-5,6-dihydroindolo[2,1-a]isoquinolines have been shown to inhibit the growth
of human mammary carcinoma cells by an unknown mode of action. One of the possible
molecular targets is the tubulin system which is involved in cell division. A number of 5,6-
dihydroindolo[2,1-a]isoquinolines with methoxy or hydroxy groups in positions 3, 9, and/or 10
and various functional groups such as formyl, acetyl, cyano, alkylimino, and alkylamino in
position 12 were synthesized and evaluated for both inhibition of tubulin polymerization and
cytostatic activity in MDA-MB 231 and MCF-7 human breast cancer cells. In the tubulin
polymerization assay, only hydroxy derivatives were active, whereas both the hydroxy
derivatives and some of the methoxy compounds inhibited cell growth. In order to establish a
correlation between the inhibition of tubulin polymerization and cytostatic activity in the
hydroxy series, two of the most active racemates were separated into the enantiomers. In
both assays, the relative potencies of the hydroxy derivatives were in a similar order. Highest
activity was found for the (+)-isomers of 6-propyl- (6b) and 6-butyl-12-formyl-5,6-hydro-3,9-
dihydroxyindolo[2,1-a]isoquinoline (6c) with IC50 values of 11 ( 0.4 and 3.1 ( 0.4 µM,
respectively, for the polymerization of tubulin at 37 °C (colchicine: 2.1 ( 0.1 µM). The active
hydroxy derivatives displaced 40-70% of [3H]colchicine from its binding site in the tubulin at
concentrations 10-fold higher than that of colchicine. The data suggest that hydroxy-substituted
indolo[2,1-a]isoquinolines bind to the colchicine-binding site and inhibit the polymerization of
tubulin. This action can be assumed to be responsible for the cytostatic activity of the hydroxy
derivatives and might also contribute to the antitumor effect of the corresponding methyl ethers.
Ch a r t 1. Inhibitors of Tubulin Polymerization
In a previous paper we reported on the cytostatic
activities and endocrine properties of a series of acetoxy-
substituted 6-alkyl-12-formyl-5,6-dihydroindolo[2,1-a]-
isoquinolines.1 Although these compounds bind to the
estrogen receptor (ER), they did not show a preference
for ER-positive cells when tested for cytostatic activity.
IC50 values obtained with estrogen-sensitive human
MCF-7 breast cancer cells were rather similar to those
obtained with hormone-independent MDA-MB 231 mam-
mary carcinoma cells. One of the interesting features
of these derivatives was the stereospecificity of the
antitumor effect, which was mainly associated with the
dextrorotary form.1 This specific activity prompted us
to search for the molecular targets of these agents. In
our early studies we failed to demonstrate an interaction
with the DNA, the most prominent target molecule for
cytostatic drugs. Other cellular structures of interest
comprise the signal transduction machinery and the
tubulin system. Preliminary studies with EGF-recep-
tor-associated protein tyrosin kinases (PTK) revealed
no activity.
The structures of the compounds which inhibit tubu-
lin polymerization exhibit a great diversity which sug-
gests different binding sites at the dimer formed by R-
and â-tubulin. The only well-defined pharmacophor for
the inhibition of tubulin polymerization is the 3,4,5-
trimethoxyphenyl group linked to another aromatic
system.2-9 It is found in colchicin,10 podophyllotoxin,11
steganacin,12 andsin its simplest formscombretastatin
A-413 (Chart 1). Other simple molecules comprise
heterocyclic systems such as 2-phenylquinolinone,14,15
† Institut fu¨r Pharmazie.
‡ Institut fu¨r Organische Chemie.
X Abstract published in Advance ACS Abstracts, September 15, 1997.
S0022-2623(97)00177-5 CCC: $14.00 © 1997 American Chemical Society

Inhibition of Tubulin Polymerization
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3525
Sch em e 1a
a
(A) NEt3; (B) 1. POCl3, 2. NaOH, 3. NaBH4; (C) 1. NaH/DMSO, 2. Pd/C (10%); (D) BBr3.
Sch em e 2a
a
(A) POCl3/DMF; (B) BBr3; (C) I2.
2-styrylquinazolin-4-one,16,17 1-deaza-7,8-dihydropteri-
dine,18 and steroids such as estramustine and 2-meth-
oxyestradiol.19,20 Different binding sites have to be
assumed for the complex structures of the vinca alka-
loids21 and maitansin,22 though an overlap might be
possible. The knowledge that estradiol disrupts micro-
tubuli23 and the known inhibitory effect of estradiol
derivatives on tubulin polymerization19,20 prompted us
to consider an interference with the formation of mi-
crotubuli as a possible mode of cytostatic action for the
indoloisoquinolines.
An appropriate method for studying the effect of drugs
on polymerization and depolymerization of tubulin is
turbidimetry. Microtubuli and R/â-tubulin dimers form
a dynamic temperature-dependent equilibrium with
preference for the polymer at 37 °C. The concentration
of polymer can be measured spectroscopically due to the
reflection of light by the microtubuli. In this study we
compared the inhibition of tubulin polymerization at 37
°C with the cytostatic activity of a variety of C-12-
modified indolo[2,1-a]isoquinolines (Chart 1) in two
human breast cancer cell lines. The most active com-
pounds were analyzed for their ability to displace
colchicine from its binding site.
Ch em istr y
The route to the tetracyclic skeleton of the 5,6-
dihydroindolo[2,1-a]isoquinoline, as outlined in Scheme
1, has been described previously.1 For this study only
derivatives with an oxygen function in position 3 and
one or two oxygen functions in the indole part (positions
9 and/or 10) were considered. These derivatives have
been shown to exert the strongest cytostatic effects when
a propyl or butyl group was linked to position 6.
The structural variations presented here concerned
the functional group in position 12 and the conversion
of the methoxy derivatives into the free phenols (Scheme
2). The formyl group in 6-butyl-12-formyl-5,6-dihydro-
3,9-dimethoxyindolo[2,1-a]isoquinoline (5c) and 6-butyl-
12-formyl-5,6-dihydro-3,9,10-trimethoxyindolo[2,1-a]i-

3526 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Goldbrunner et al.
Sch em e 3a
a
(A) HONH3Cl/Na2CO3; (B) Ac2O/pyridine; (C) BBr3; (D) HOAc; (E) NaBH4.
soquinoline (5e), respectively, was converted into the
nitrile (11a ,b) via the oxime (10a ,b).
those of the corresponding methyl ethers. When the
dose-response curves of some of the more potent
hydroxy derivatives were plotted, deviations from the
sigmoid curve were observed with a more or less
pronounced minimum in the submicromolar range and
the normal decline above 1 µM. In the case of 6b and
12a the minimum was as low as 25-30% T/C at 5 ×
10-7 M. This biphasic course of the curve was also
found for 2-methoxyestradiol and was confirmed in
additional experiments.
Compound 5c reacted with methylamine and benz-
ylamine to produce the respective imines 13a ,b, which
gave the corresponding amines 14a ,b upon treatment
with sodium boron hydride (Scheme 3). As an alterna-
tive to the formyl group, the acetyl group was introduced
into the indolo[2,1-a]isoquinoline system by treatment
with acetic acid anhydride in the presence of iodine to
give the 12-acetyl derivative 8. All of the methoxy
compounds except the imines 13 and amines 14 were
converted to the free phenols by treating them with
boron tribromide. Direct reaction of the phenolic alde-
hyde 6c with benzylamine led to the imine 7 with
unprotected hydroxy groups.
When the cytostatic effects of the hydroxy derivatives
were compared with the inhibition of tubulin polymer-
ization, some agreement was observed though the
concentration in the tubulin polymerization assay was
considerably higher. The discrepancy between concen-
trations required for cytostatic action and those for the
inhibition of tubulin polymerization has been noticed
in many other classes of antimitotic agents.4,7,8,24 The
most active hydroxy compounds were the formyl deriva-
tives 6b,d and the nitrile 12a with IC50 values below
10-6 M. The benzylimine of 6c (7) showed an inhibitory
effect similar to that of the parent compound which
might be due to a hydrolysis reaction under the condi-
tions of the assay.
Biologica l Resu lts a n d Discu ssion
The aim of this study was the identification of the
molecular target which is involved in the cytostatic
action of 12-substituted 5,6-dihydroindolo[2,1-a]isoquin-
olines. Since preliminary investigations revealed nei-
ther an interaction with the DNA nor an interference
with the signal transduction machinery, we considered
the tubulin or its polymeric structure, the microtubuli,
as the potential site of action. The tubulin used in this
study was isolated from fresh calf brains by several
centrifugation steps at two different temperatures: 37
°C to polymerize tubulin to microtubuli and 2 °C for
depolymerization. Two cycles of polymerization/depo-
lymerization were usually sufficient to get electro-
phoretically pure tubulin that was still associated with
MAPs (microtubuli-associated proteins). In the initial
experiments, all compounds of this study were evaluated
for their inhibitory activity on tubulin polymerization
in a standard concentration of 40 µM (Table 1). Strong
inhibition was only observed for the hydroxy derivatives
but not for the corresponding methyl ethers. Among
the free phenols, the 12-unsubstituted tetracycle 4, the
6-ethyl derivative 6a , and the 12-acetyl compound 9
were only weak inhibitors.
Similar results were obtained with estrogen-sensitive
MCF-7 breast cancer cells which were used as an
additional model for the evaluation of the phenolic
derivatives because of their affinity for the estrogen
receptor. The relative binding affinities for the calf
uterine estrogen receptor were in the range of 0.5-2.0%
of estradiol. At a concentration of 1 µM, the 6-ethyl (6a )
and the 12-acetyl (9) derivatives as well as the trihy-
droxy compounds 6e and 12b were inactive, whereas
all of the other hydroxy derivatives completely blocked
cell proliferation. For the majority of the tested com-
pounds no marked differences between both cell lines
were detected. Thus, an estrogen receptor-mediated
action in the estrogen receptor-positive MCF-7 cells is
unlikely.
Both methoxy derivatives and free phenolic com-
pounds were tested for cytostatic activity in human
MDA-MB 231 breast cancer cells which lack estrogen
receptors. In this assay the methoxy compounds inhib-
ited cellular growth to a varying degree. Lowest IC50
values were found for both nitriles (11a ,b ∼ 3 µM) and
the methylamino derivative 14a (2.1 µM) (Table 1). The
cytostatic activities of the phenolic compounds exceeded
In a previous paper,1 we reported on the stereospeci-
ficity of the antitumor effect of the acetates of 6b,c. This
observation prompted us to separate the racemic mix-
tures of the hydroxy derivatives 6b,c by liquid chroma-
tography to obtain the pure enantiomers (+)-6b, (-)-
6b and (+)-6c, (-)-6c, respectively. These compounds
together with the racemates and the racemic mixtures
of 6d ,e, and 12a ,b were tested at several concentrations

Inhibition of Tubulin Polymerization
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3527
Ta ble 1. Effect of Various Methoxy- and Hydroxy-Substituted 6-Alkyl-5,6-dihydroindolo[2,1-a]isoquinolines on Tubulin
Polymerization and the Growth of Human Mammary Carcinoma Cells
IC50 [µM]
compd
3e
5b
5c
5d
5e
8
10a
11a
11b
13a
13b
14a
14b
4
6a
6b
6c
6d
6e
7
9
12a
12b
R1
R2
R3
R4
R5
ITPa (%)
MDAb
MCF-7c
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
n-C4H9
n-C3H7
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
C2H5
n-C3H7
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
n-C4H9
OCH3
OCH3
OCH3
H
OCH3
H
H
41
17
11
10
1
>10
>10
CHO
CHO
CHO
CHO
COCH3
H
8.7
OCH3
OCH3
H
>10
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
9.0
5
>10
>10
H
H
CHdNOH
17
16
32
3
16
9
27
33
11
93
100
81
100
93
24
100
79
100
CN
CN
3.1
OCH3
H
2.7
7.8
5.7
2.1
7.0
CHdNCH3
CHdNCH2C6H5
CH2NHCH3
CH2NHCH2C6H5
H
H
H
H
OH
H
>10
>10
2.5
OH
OH
OH
CHO
CHO
CHO
CHO
2.8
0.3
1.4
0.2
3.2
2.1
2.2
0.3
3.4
0.03
H
H
0.29
0.22
0.65
>10
0.26
2.5
H
OH
OH
H
OH
OH
OH
OH
CHO
CHdNCH2C6H5
COCH3
CN
H
H
0.54
2.7
OH
OH
OH
CN
colchicine
tamoxifen
0.15
a
Inhibition of tubulin polymerization by a 40 µM solution of test compounds, measured after 20 min at 37 °C. Mean values of two
b
independent experiments. Full details are presented in the text. Inhibition of the growth of MDA-MB 231 human breast cancer cells
after incubation for 4 days, determined by measuring optical densities after crystal violet staining of vital cells. c Inhibition of the growth
of estrogen-sensitive MCF-7 human breast cancer cells after incubation for 8 days, determined by measuring optical densities after crystal
violet staining of vital cells. Only derivatives with free hydroxy groups and tamoxifen as reference drug were tested.
exclusively the (+)-isomers with IC50 values of 11 and
3.1 µM, respectively, were responsible for the inhibition
of tubulin polymerization of the racemates 6b,c. The
value for (+)-6c was close to those of the reference drugs
colchicine (2.1 µM) and podophyllotoxin (1.2 µM). The
differences in activity between the enantiomers were
paralleled by the results obtained with the acetates in
MDA-MB 231 cells. The lack of sufficient quantities of
the pure enantiomers of the phenols did not allow us to
use the free hydroxy derivatives for the determination
of cytostatic activity. Significant differences in cyto-
static activity between hydroxy derivatives and their
actetates however have not been noted for several 5,6-
dihydroindolo[2,1-a]isoquinolines (data not shown) or
2-phenylindoles25 when both types of compounds were
tested in vitro for growth inhibition of MDA-MB 231 and
MCF-7 cells, respectively. The values for the reference
drugs colchicine and podophyllotoxin were close to those
reported in the literature for their inhibitory effects on
tubulin polymerization (colchicine 1.9;14 podophyllotoxin
2.1 ( 0.1,7 1.3 ( 0.614).
Inhibition of tubulin polymerization can be considered
as the consequence of the binding interaction with the
R/â-tubulin dimer though other mechanisms such as
interactions with magnesium ions or GTP are conceiv-
able. One of the obvious sites is that at which colchicine
and a number of other agents with two aromatic
domains bind. Thus, we tested the enantiomers and
F igu r e 1. Effect of various concentrations of 5,6-dihydroin-
dolo[2,1-a]isoquinoline 6e on the polymerization of calf brain
tubulin. UV absorption was recorded for 20 min after the
temperature had been switched from 2 to 37 °C. Control curve
represents unaffected tubulin polymerization.
for their inhibitory effect on tubulin polymerization
(Figure 1, Table 2). Characteristic polymerization
curves were obtained for all of the compounds with
plateaus on different levels (Figure 1). From the
calculated data it became evident that mainly or even

3528 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Goldbrunner et al.
Ta ble 2. Inhibitory Effects of Racemic 5,6-Dihydroindolo[2,1-a]isoquinolines and Enantiomers on Tubulin Polymerization, Colchicine
Binding, and Cell Growth
ICGc (IC50, µM)
compd
ITPa (IC50, µM, ( SD)
ICBb (%)
R1,R3(4) ) OH
R1,R3 ) OAc
(()-6b
(+)-6b
(-)-6b
(()-6c
(+)-6c
(-)-6c
6d
6e
12a
12b
colchicine
19 ( 1 (16 ( 2d)
40 (33d)
53
27
0.3
nd
nd
1.4
nd
nd
0.45d
0.35d
5.5d
11 ( 0.4
>70
8.9 ( 0.3 (4.9 ( 0.1 at 26 °C)
67
0.32d
0.15d
0.78d
3.1 ( 0.4
69e
22
>60
19 ( 3
nd
57
48
54
0.2
3.2
0.3
19 ( 1
13 ( 2
25 ( 1
3.4
2.1 ( 0.1 (1.7 ( 0.05 at 26 °C)
1.2 ( 0.1
0.030
0.026
0.011
podophyllotoxin
vinblastine
2-methoxyestradiol
89
-64
44
0.7 ( 0.1
60 ( 2 (12 ( 0.5 at 26 °C)
0.25 and ∼5f
a
Inhibition of tubulin polymerization: IC50 values were determined after 20 min at 37 °C. Mean values of three independent experiments.
Full details are presented in the text. Inhibition of colchicine binding. Reaction mixtures contained tubulin, 1 µM [3H]colchicine, and 10
b
µM inhibitor and were incubated for 30 min at 37 °C prior to analysis. Mean of two independent experiments. c Inhibition of cell growth.
d
The IC50 values were determined with MDA-MB 231 breast cancer cells. Mean of two independent experiments. Values of the respective
diacetates whose syntheses are described in ref.1 e 30% inhibition for equimolar concentrations (1 µM) of colchicine and inhibitor. f Biphasic
dose-response curve with maximum inhibition (91%) at 1 and >10 µM.
some of the racemic mixtures for their capability of
displacing [3H]colchicine from its binding site. At
concentrations 10-fold higher than that of colchicine, the
tubulin-bound radioactivity was decreased substan-
tially. The differences between the optical isomers,
though also observed in this experiment, were less
pronounced than in the other assays. Podophyllotoxin
displaced 89% of colchicine from its binding site when
dosed 10-fold higher and 69% in equimolar concentra-
tion. These values are similar to those reported by other
authors.16 Vinblastine increased the amount of colchi-
cine bound to tubulin due to a stabilization of the
binding.26
The comparison with 2-methoxyestradiol was of par-
ticular interest in this study because this compound
shares the binding affinity for the estrogen receptor with
the indoloisoquinoline system and has been reported to
inhibit tubulin polymerization by interacting with the
colchicine-binding site.19 The latter effect was only
marked at suboptimal conditions at a polymerization
temperature of 26 °C and was rather small at 37 °C.
We made the same observation when we tested 2-meth-
oxyestradiol at both temperatures. The values of other
compounds such as colchicine or 6c were also influenced
by the temperature but to a smaller extent (Table 2).
Similar results were obtained in a series of diaryl
compounds when tested at 30 and 37 °C.6 The binding
affinity of methoxyestradiol for the colchicine-binding
site was somewhat lower than that observed for the
most active indoloisoquinolines and was consistent with
that reported in the literature (34% inhibition versus
38%27).
which 5,6-dihydroindolo[2,1-a]isoquinolines exert their
antitumor activity and the improvement of cytostatic
potency of this class of compounds by modifying the
substituent at C-12. One of the molecular targets that
we considered for the action of these compounds was
the tubulin system. When we evaluated the effect of
various indoloisoquinoline derivatives on the polymer-
ization of tubulin, only hydroxy derivatives proved to
be inhibitors with variable potencies whereas none of
the methoxy derivatives strongly interfered with the
formation of microtubuli. This result was unexpected
because most of the known antimitotic compounds are
characterized by the presence of aromatic methoxy
groups as exemplified by colchicine and combretastatin
A-4 (Chart 1). Since the synthetic procedures applied
did not allow the preparation of derivatives with only
one ether function cleaved, it was not possible to study
mixed hydroxy/methoxy compounds. Thus, it remains
unclear whether one or two phenolic groups are required
for strong inhibition of tubulin polymerizaton.
The structural alterations concerned mainly the sub-
stituent at C-12 of the tetracyclic indoloisoquinoline
system. The replacement of the formyl group by other
functional groups such as acetyl, alkylimino, alky-
lamino, and cyano improved neither the cytostatic
activity nor the inhibitory effect on tubulin polymeri-
zation. The alkylimino derivatives appeared to function
as prodrugs giving the same results as the parent formyl
compound. A second hydroxy group in the indole moiety
brought no advantage.
When the cytostatic activities of the hydroxy deriva-
tives were compared with their ability of inhibiting
tubulin polymerization, a similar order of relative
potencies was observed. The average IC50 values for
these two effects, however, were quite different. The
concentrations for the inhibition of cell growth were 2
orders of magnitude lower than those for the cell-free
experiments on tubulin polymerization. This difference
however seems to be a general feature of antimitotic
drugs because it has been reported in several pa-
pers4,7,8,24,28 and might reflect the differences in the
experimental design. In the cellular assay, living cells
are treated at 37 °C with the drug, whereas the
turbidimetric determinations are performed with a
When we determined the cytostatic activity of 2-meth-
oxyestradiol in MDA-MB 231 cells, we noticed a biphasic
course of the dose-response curve. Maximum growth
inhibition was observed at a concentration of 1 µM. At
lower and higher concentrations the inhibitory effect
was diminished. Possibly, a protective effect conteracts
the inhibition at concentrations between 1 and 10 µM.
This unusual reaction of the MDA-MB cells, which was
also observed for 6a and 12a , was confirmed by repeat-
ing these experiments several times.
The investigations reported in this paper were aimed
in two directions: the elucidation of the mechanism by

Inhibition of Tubulin Polymerization
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3529
(m, 1H, -CH-NH-), 5.20 (d, br, 3J ) 9 Hz, 1H, -NH-), 6.52-
7.26 (m, 4H, Ar-H), 6.75 (s, 1H, Ar-H), 7.01 (s, 1H, Ar-H). Anal.
(C23H30BrNO4) C, H, N.
preparation of isolated tubulin and involve a depolym-
erization step followed by the temperature-dependent
polymerization. It is conceivable that small quantities
of an antimitotic compound can interact with a few
essential sites in the microtubuli leading to disruption
and preventing the formation of the mitotic spindle.
Therefore, we have planned to analyze the antibody-
labeled microtubuli by fluorescence microscopy in the
presence and absence of antimitotic drugs. Maybe, it
is possible to detect cellular changes at concentrations
significantly lower than those required for inhibition of
polymerization.
In this study, we found a distinct difference between
the hydroxy-substituted indoloisoquinolines and the
methoxy derivatives with cytostatic activity such as the
two nitriles and the methylamino derivative. Though
compounds of both series possess considerable cytostatic
activity, only the phenolic compounds inhibit the po-
lymerization of tubulin under the conditions of the
turbidimetry assay. Several reasons can account for
this apparent difference: (i) The methoxy derivatives
inhibit cellular growth by a mode of action different from
that of the phenolic compounds although both series
share the same tetracyclic skeleton. (ii) The increased
lipophilicity of the methyl ether facilitates the penetra-
tion of the compounds into the tumor cells which might
increase cytostatic activity. (iii) A different kind of
interaction with the tubulin takes place which does not
prevent polymerization. An interesting example for the
latter effect is curacin A, a newly isolated lipid natural
product that binds to the colchicine site of tubulin and
inhibits mitosis.24 It was shown to interfere with the
polymerization reaction in a rather unusual manner
giving rise only to partial inhibition even at high
concentrations.29 Under these aspects, further studies
with different techniques are warranted to clarify
whether the methoxy-substituted indolo[2,1-a]isoquino-
lines act via the tubulin system or an alternate mech-
anism.
1-(2-Br om o-4,5-d im et h oxyb en zyl)-3-b u t yl-1,2,3,4-t et -
r a h yd r o-6-m eth oxyisoqu in olin e (2). Under N2, 66 mmol
of POCl3 was added to a solution of the acetamide 1 (23 mmol)
in 35 mL of dry MeCN. After heating for 3 h under reflux,
the mixture was poured into ice-water. With cooling, the
mixture was made alkaline by addition of 40% aqueous NaOH.
The aqueous solution was extracted with CH2Cl2. The com-
bined organic layers were washed with water and saline and
dried (Na2SO4). After evaporation of the solvent, the crude
product was dried under high vacuum and dissolved in 100
mL of dry MeOH. After cooling to 0 °C, sodium borohydride
(73 mmol) was added in small portions. Stirring was contin-
ued at room temperature for 1 h followed by heating under
reflux for 1 h. After evaporation of the solvent, CH2Cl2 and
water were added. The organic layer was separated and the
aqueous one extracted with CH2Cl2. The combined organic
layers were washed with saturated NaCl solution and dried
(Na2SO4). The residue obtained after evaporation of the
solvent was purified by chromatography (SiO2; CH2Cl2/Et2O,
5:1). The product was obtained as a colorless oil (42%): 1H
3
NMR (CDCl3) δ 0.87 (t, J ) 7 Hz, 3H, -CH2-CH3), 1.18-1.50
(m, 6H, -(CH2)3-CH3), 1.55 (s, br, 1H, -NH-), 2.52-2.79 (m, 4H,
2
3
-CH2-CH-, -CH-H), 3.61, 3.66 (dd, J ) 14 Hz, J ) 3 Hz, 1H,
-CH-H), 3.79 (s, 3H, -OCH3), 3.83 (s, 3H, -OCH3),3.88(s, 3H,-
OCH3), 4.27 (d, 3J ) 8 Hz, 1H, Ar-CH-NH), 6.64 (d, 3J ) 9 Hz,
1H, Ar-H), 6.75, 6.79 (dd, 3J ) 9 Hz, 4J ) 3 Hz, 1H, Ar-H),
3
6.89 (s, 1H, Ar-H), 7.07 (s, 1H, Ar-H), 7.34 (d, J ) 9 Hz, 1H,
Ar-H). Anal. (C23H30BrNO3) C, H, N.
6-Bu tyl-5,6-d ih yd r o-3,9,10-tr im eth oxyin d olo[2,1-a ]iso-
qu in olin e (3e). A solution of (bromobenzyl)tetrahydroiso-
quinoline 2 (9.5 mmol) in 37 mL of DMSO was added to a
solution of sodium (methylsulfinyl)methanide prepared from
67 mmol of NaH (80% in oil dispersion) and 37 mL of DMSO.
After stirring for 18 h, the mixture was poured into 300 mL of
water containing an excess of NH4Cl and extracted with CH2-
Cl2. The organic layer was washed with water and saline.
After drying (Na2SO4) and evaporation of the solvent, an oil
was obtained. Chromatography (SiO2; CH2Cl2) yielded a
mixture of dihydro- and tetrahydroindoloisoquinolines which
was not separated but directly converted into the dihydro
derivative.
Under N2, the mixture of dihydro- and tetrahydroindoloiso-
quinolines (2.5 g) and Pd/C (10%, 0.90 g) was mixed thoroughly
in a round bottom flask, which was then placed in a hot oil
bath. The temperature was kept at 120 for 4 h. After cooling,
the mixture was dissolved in CHCl3 and filtered. The solvent
was evaporated and the residue crystallized from EtOH. After
recrystallization colorless crystals (45%) were obtained: mp
Exp er im en ta l Section
Melting points were determined on a Bu¨chi 510 apparatus
and are uncorrected. Elemental analyses were performed by
Mikroanalytisches Laboratorium, University of Regensburg,
and were within (40% of the calculated values except where
noted. NMR spectra were obtained on a Bruker AC-250
spectrometer with TMS as internal standard and are consis-
tent with the assigned structures. Mass spectra were recorded
on a Varian MAT 311A spectrometer. The syntheses of the
6-alkyl-5,6-dihydro-3,9(10)-dimethoxyindolo[2,1-a]isoquino-
lines 3a -d and their 12-formyl derivatives 5a -d have been
described previously.1
3
137-139 °C; 1H NMR (CDCl3) δ 0.82 (t, J ) 7 Hz, 3H, -CH2-
2
CH3), 1.19-1.59 (m, 6H, -(CH2)3-CH3), 2.93, 2.99 (dd, J AB
)
3
2
16 Hz, J AX ) 1 Hz, 1H, HA-5), 3.33, 3.38 (dd, J AB ) 16 Hz,
3J BX ) 6 Hz, 1H, HB-5), 3.84 (s, 3H, -OCH3), 3.93 (s, 3H,
-OCH3), 3.96 (s, 3H, -OCH3), 4.50-4.58 (m, 1H, H-6), 6.62 (s,
1H, H-8), 6.77 (d, 4J ) 2 Hz, 1H, H-4), 6.80 (s, 1H, H-11), 6.81,
6.85 (dd, J ) 2 Hz, J ) 9 Hz, 1H, H-2), 7.06 (s, 1H, H-12),
7.60 (d, J ) 9 Hz, 1H, H-1). Anal. (C23H27NO3) C, H, N.
4
3
3
(2-Br om o-4,5-d im eth oxyp h en yl)-N-[1-(3-m eth oxyp h e-
n yl)h ex-2-yl]acetam ide (1). Under N2, a solution of 2-bromo-
4,5-dimethoxyphenylacetic acid chloride (0.10 mol) in 110 mL
of dry CH2Cl2 was added dropwise to a stirred solution of
2-amino-1-(3-methoxyphenyl)hexane (0.10 mol) and triethy-
lamine (0.10 mol) in 250 mL of dry CH2Cl2. Stirring was
continued at room temperature for 1 h. The reaction mixture
was poured into ice-water, acidified with 2 N HCl, and
extracted with CH2Cl2. The combined organic layers were
washed with NaHCO3 solution and water and dried (Na2SO4).
After evaporation of the solvent the crude product was purified
by chromatography (SiO2; CH2Cl2/Et2O, 7:1). The product was
crystallized from Et2O/EtOAc (1:1) to afford colorless crystals
(23%): mp 112-114 °C; 1H NMR (CDCl3) δ 0.86 (t, 3J ) 7 Hz,
6-Bu tyl-5,6-d ih yd r o-3,9,10-tr ih yd r oxyin d olo[2,1-a ]iso-
qu in olin e (4). Under N2, 5.5 mmol of BBr3 in 5 mL of dry
CH2Cl2 was added slowly to a solution of the trimethoxy
derivative 3e in 15 mL of dry CH2Cl2 at -50 °C. After
addition, stirring was continued for 30 min at -50 °C and 18
h at room temperature. The mixture was carefully hydrolyzed
by adding saturated NaHCO3 solution. After addition of 60
mL of EtOAc, the mixture was vigorously stirred for 15 min.
After separation of the layers the aqueous phase was extracted
several times with EtOAc. The combined organic layers were
washed with water and saline and dried (Na2SO4). After
evaporation of the solvent in vacuo, the product was purified
by chromatography (SiO2; CH2Cl2/Et2O, 2:1) to give off-white
crystals (88%): mp 183-185 °C; 1H NMR (CDCl3) δ 0.81 (t, 3J
) 7 Hz, 3H, -CH2-CH3), 1.16-1.51 (m, 6H, -(CH2)3-CH3), 2.97,
3
3H, -CH2-CH3), 1.04-1.49 (m, 6H, -(CH2)3-CH3), 2.72 (d, J )
2
3
7 Hz, 2H, -CH2-CH-), 3.59 (s, 2H, CO-CH2-), 3.76 (s, 3H,
-OCH3), 3.84 (s, 3H, -OCH3), 3.88 (s, 3H, -OCH3), 4.05-4.22
3.03 (dd, J AB ) 16 Hz, J AX ) 1 Hz, 1H, HA-5), 3.22, 3.29 (dd,
3
2J AB ) 16 Hz, J BX ) 6 Hz, 1H, HB-5), 4.55 (m, 1H, H-6), 6.50

3530 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Goldbrunner et al.
(s, 1H, vinyl-H), 6.74 (s, 1H, OH) 6.75 (s, 1H, Ar-H), 6.78 (s,
1H, Ar-H), 6.85 (s, 1H, Ar-H), 7.46 (s, 1H, OH), 7.50-7.63 (m,
2H, Ar-H), 8.39 (s, 1H, OH). Anal. (C20H21NO3) C, H, N.
6-Bu tyl-12-for m yl-5,6-d ih yd r o-3,9,10-tr im eth oxyin d o-
lo[2,1-a ]isoqu in olin e (5e). Dry DMF (1.9 mL) was added
slowly to 29 mmol of POCl3 at 10-20 °C under N2. A solution
of 3e (2.9 mmol) in 18 mL of dry DMF was added slowly to
keep the temperature below 35 °C. Stirring was continued
for 1 h at room temperature. The mixture was then poured
into ice-water and made alkaline by addition of NaOH (40%).
The solution was extracted with CH2Cl2. The organic layer
was washed with water and saline and dried (Na2SO4). The
solvent was removed in vacuo and the residue purified by
chromatography (SiO2; CH2Cl2/Et2O, 5:1) to give a greenish
amorphous material (76%): mp 78-80 °C; 1H NMR (CDCl3) δ
1H NMR (DMSO-d6) -CH3), 1.15-1.45 (m, 6H, -(CH2)3-CH3),
2
3.01 (dd, 2J AB/3J AX ) 16/1 Hz, 1H, -CH-H, HA-5), 3.24 (dd, 2J AB
/
3J BX ) 16/6 Hz, 1H, -CH-H, HB-5), 4.74 (m, 1H, -CH-, H-6),
6.73-6.86 (m, 3H, Ar-H), 7.39 (d, J ) 9 Hz, 1H, Ar-H), 7.64
(d, J ) 2 Hz, 1H, Ar-H), 7.84 (d, J ) 8 Hz, 1H, Ar-H), 9.12 (s,
1H, -OH), 10.08 (s, 1H, -OH), 10.25 (s, 1H, -CHO); IR (KBr)
3265, 1615 cm-1. Anal. (C21H21NO3) C, H, N.
6-Bu tyl-12-for m yl-5,6-d ih yd r o-3,9,10-tr ih yd r oxyin d o-
lo[2,1-a ]isoqu in olin e (6e): prepared in 70% yield from 5e
by a method similar to that described for 4. Crystallization
from CHCl3 gave light-green crystals; mp 244-246 °C; 1H-
3
NMR (CDCl3) δ 0.78 (t, J ) 7 Hz, 3H, -CH2-CH3), 1.15-1.41
2
3
(m, 6H, -(CH2)3-CH3), 2.95, 3.01 (dd, J AB ) 16 Hz, J AX ) 1
2
3
Hz, HA-5), 3.16, 3.23 (dd, J AB ) 16 Hz, J BX ) 6 Hz, 1H, HB-
5), 4.61 (m, 1H, H-6), 6.77-6.83 (m, 2H, H-2, H-4), 6.90 (s,
1H, H-8), 7.63 (s, 1H, H-11), 7.76 (d, 3J ) 8 Hz, 1H, H-1), 8.85
(s, 1H, OH), 8.95 (s, 1H, OH), 9.97 (1H, OH), 10.20 (s, 1H,
-CHdO); IR (KBr) 1615 cm-1. Anal. (C21H21NO4) C, H, N.
12-[(N-Ben zylim in o)m et h yl]-6-b u t yl-5,6-d ih yd r o-3,9-
d ih yd r oxyin d olo[2,1-a ]isoqu in olin e (7). After addition of
ca. 50 mg of CaSO4 and 0.5 mL of AcOH, the solution of 3c
(200 mg, 0.55 mmol) in a mixture of 3 mL of benzylamine and
7 mL of EtOH was stirred at room temperature under nitrogen
atmosphere overnight. After filtration, the solvent and excess
of amine were removed in vacuo. The residue was diluted with
50 mL of water and extracted with Et2O. The combined
organic layers were washed twice with water and dried (Na2-
SO4). After evaporation of the solvent a crystalline product
was obtained which was recrystallized from Et2O to give dark-
red crystals (97%); dec >110 °C; 1H NMR (acetone-d6) 2)3-CH3),
1.22-1.55 (m, 6H, -(CH2)3-CH3), 3.05 (dd, 2J AB/3J AX ) 15/1 Hz,
3
0.84 (t, J ) 7 Hz, 3H, -CH2-CH3), 1.21-1.68 (m, 6H, -(CH2)3-
2
3
CH3), 3.00, 3.02 (dd, J AB ) 16 Hz, J AX ) 1 Hz, 1H, HA-5),
2
3
3.33, 3.38 (dd, J AB ) 16 Hz, J BX ) 6 Hz, 1H, HB-5), 3.89 (s,
3H, -OCH3), 3.97 (s, 3H, -OCH3), 4.01 (s, 3H, -OCH3), 4.51-
4.59 (m, 1H, H-6), 6.82 (s, 1H, H-8), 6.88 (d, 4J ) 2 Hz, 1H,
H-4), 6.91, 6.94 (dd, 3J ) 9 Hz, 4J ) 2 Hz, 1H, H-2), 7.82 (d, 3J
) 9 Hz, 1H, H-1), 7.95 (s, 1H, H-11), 10.41 (s, 1H, -CHO); IR
(KBr) 1645 cm-1. Anal. (C24H27NO4) C, H, N.
6-Eth yl-12-for m yl-5,6-dih ydr o-3,9-dih ydr oxyin dolo[2,1-
a ]isoqu in olin e (6a ). Under N2, 0.35 mL (3.6 mmol) of BBr3,
dissolved in 2 mL of dry CH2Cl2, was slowly added to a solution
of 6-ethyl-12-formyl-5,6-dihydro-3,9-dimethoxyindolo[2,1-a]i-
soquinoline (5a )1 (1.0 mmol) in 18 mL of dry CH2Cl2 at a
temperature of -50 °C. After stirring for 30 min at this
temperature, the mixture was allowed to warm up to room
temperature, and stirring was continued for 15 h. With cooling
a saturated solution of NaHCO3 was added until the vigorous
reaction ceased. After addition of 60 mL of EtOAc, stirring
was continued for 15 min followed by filtration. The layers
were separated, and the aqueous layer was extracted three
times with EtOAc. After washing with water and drying (Na2-
SO4), the solvent was removed in vacuo and the residue
purified by chromatography (SiO2; CHCl3/Et2O, 1:1). Crystal-
lization from CHCl3 afforded light-green crystals (83%): dec
>215 °C; 1H NMR (DMSO-d6) 2-CH3), 1.28-1.62 (m, 2H, -CH2-
2
1H, -CH-H, HA-5), 3.27 (dd, J AB/3J BX ) 15/6 Hz, 1H, -CH-H,
HB-5), 4.64 (m, 1H, -CH-, H-6), 4.85 (s, 2H, dN-CH2-), 6.71
(dd, J 1/2 ) 9/2 Hz, 1H, Ar-H), 6.80-6.91 (m, 3H, Ar-H), 7.19-
7.46 (m, 5H, -C6H5), 7.71 (d, J ) 8 Hz, 1H, Ar-H), 8.34 (d, J )
8 Hz, 1H, Ar-H), 8.40 (s, br, 2H, 2 × -OH), 9.02 (s, 1H, H-Cd
N-); IR (KBr) 3280, 1620 cm-1; MS (EI) m/ z 424 (M•+). Anal.
(C28H28N2O2) C, H, N.
12-Acetyl-6-bu tyl-5,6-d ih yd r o-3,9-d im eth oxyin d olo[2,1-
a ]isoqu in olin e (8). After addition of a catalytic amount of
iodine, the mixture of 335 mg (1 mmol) of 6-butyl-5,6-dihydro-
3,9-dimethoxyindolo[2,1-a]isoquinoline (1c) and 2 mL of Ac2O
was heated for 3 h at 150 °C. After cooling, ice water was
added and the mixture extracted with CH2Cl2. The combined
organic layers were washed with saturated NaHCO3 solution,
saturated NaHSO3 solution, and water. After drying (Na2SO4),
the solvent was removed and the residue chromatographed
(SiO2; CH2Cl2/EtOAc, 10:1). The solid obtained in 71% yield
was recrystallized from Et2O to give beige crystals: mp 115
°C; 1H NMR (CDCl3) δ 0.83 (t, 2-CH3), 1.19-1.57 (m, 6H,
-(CH2)3-CH3), 2.64 (s, 3H, -CO-CH3), 2.95 (dd, 2J AB/3J AX ) 15/1
Hz, 1H, -CH-H, HA-5), 3.34 (dd, 2J AB/3J BX ) 15/6 Hz, 1H, -CH-
H, HB-5), 3.88 (s, 3H, -OCH3), 3.90 (s, 3H, -OCH3), 4.53 (m,
1H, -CH-, H-6), 6.80-6.92 (m, 4H, Ar-H), 7.92 (d, J ) 9 Hz,
2
CH3), 3.02 (dd, J AB/3J AX ) 16/1 Hz, 1H, -CH-H, HA-5), 3.22
(dd, 2J AB/3J BX ) 16/6 Hz, 1H, -CH-H, HB-5), 4.66 (m, 1H, -CH-,
H-6), 6.71 (dd, J 1/2 ) 8/2 Hz, 1H, Ar-H), 6.80-6.89 (m, 3H,
Ar-H), 7.82 (d, J ) 8 Hz, 1H, Ar-H), 7.98 (d, J ) 8 Hz, 1H,
Ar-H), 9.41 (s, 1H, -OH), 10.06 (s, 1H, -OH), 10.26 (s, 1H,
-CHO); IR (KBr) 3320, 1610 cm-1. Anal. (C19H17NO3) C, H,
N.
12-F or m yl-5,6-d ih yd r o-3,9-d ih yd r oxy-6-p r op ylin d olo-
[2,1-a ]isoqu in olin e (6b): prepared in 89% yield from 12-
formyl-5,6-dihydro-3,9-dimethoxy-6-propylindolo[2,1-a]isoquin-
oline (5b)1 by a method similar to that described for 6a ; light-
green crystals; dec >225 °C; 1H NMR (DMSO-d6) 2-CH3), 1.18-
2
1.41 (m, 4H, -(CH2)2-CH3), 3.01 (dd, J AB/3J AX ) 16/1 Hz, 1H,
2
-CH-H, HA-5), 3.23 (dd, J AB/3J BX ) 16/6 Hz, 1H, -CH-H, HB-
5), 4.69 (m, 1H, -CH-, H-6), 6.74 (dd, J 1/2 ) 8/2 Hz, 1H, Ar-H),
6.80-6.90 (m, 3H, Ar-H), 7.83 (d, J ) 8 Hz, 1H, Ar-H), 8.00
(d, J ) 8 Hz, 1H, Ar-H), 9.40 (s, 1H, -OH), 10.05 (s, 1H, -OH),
1H, Ar-H), 8.05 (d, J ) 9 Hz, 1H, Ar-H); IR (KBr) 1630 cm-1
.
Anal. (C24H27NO3) C, H, N.
12-Acetyl-6-bu tyl-5,6-d ih yd r o-3,9-d ih yd r oxyin d olo[2,1-
a ]isoqu in olin e (9): prepared in 67% yield from 8 by a method
similar to that for 6a ; purification by chromatography (SiO2;
CH2Cl2/Et2O, 1:1) gave light-green amorphous material; 1H
NMR (DMSO-d6) 2-CH3), 1.17-1.36 (m, 6H, -(CH2)3-CH3), 2.51
10.26 (s, 1H, -CHO); IR (KBr) 3310, 1610 cm-1. Anal. (C20H19
NO3) H, N; C: calcd, 74.75; found, 74.29.
-
6-Bu tyl-12-for m yl-5,6-dih ydr o-3,9-dih ydr oxyin dolo[2,1-
a ]isoqu in olin e (6c): prepared in 85% yield from 6-butyl-12-
formyl-5,6-dihydro-3,9-dimethoxyindolo[2,1-a]isoquinoline (5c)1
by a method similar to that decribed for 6a ; light-green
2
(s, 3H, -CO-CH3), 2.96 (dd, J AB/3J AX ) 15/1 Hz, 1H, -CH-H,
2
HA-5), 3.19 (dd, J AB/3J BX ) 15/6 Hz, 1H, -CH-H, HB-5), 4.62
1
crystals; mp 144-146 °C; H NMR (DMSO-d6)2-CH3), δ (t, J
(m, 1H, -CH-, H-6), 6.67-6.84 (m, 4H, Ar-H), 7.72 (d, J ) 9
Hz, 1H, Ar-H), 7.83 (d, J ) 9 Hz, 1H, Ar-H), 9.29 (s, 1H, -OH),
) 7 Hz, 3H, -CH2-CH3), 1.18-1.46 (m, 6H, -(CH2)3-CH3), 3.01
(dd, 2J AB/3J AX ) 16/1 Hz, 1H, -CH-H, HA-5), 3.22 (dd, 2J AB/3J BX
) 16/6 Hz, 1H, -CH-H, HB-5), 4.68 (m, 1H, -CH-, H-6), 6.73
(dd, J 1/2 ) 8/2 Hz, 1H, Ar-H), 6.80-6.89 (m, 3H, Ar-H), 7.83
(d, J ) 8 Hz, 1H, Ar-H), 7.99 (d, J ) 8 Hz, 1H, Ar-H), 9.40 (s,
1H, -OH), 10.04 (s, 1H, -OH), 10.25 (s, 1H, -CHO); IR (KBr)
3300, 1610 cm-1. Anal. (C21H21NO3) C, H, N.
9.88 (s, 1H, -OH); IR (KBr) 3305, 1625 cm-1. Anal. (C22H23
NO3) C, H, N.
-
6-Bu tyl-5,6-d ih yd r o-12-[(N-h yd r oxyim in o)m eth yl]-3,9-
d im eth oxyin d olo[2,1-a ]isoqu in olin e (10a ). A solution of
400 mg (5.8 mmol) of hydroxylamine hydrochloride in 2 mL of
water was added slowly to a solution of 200 mg (0.55 mmol)
of 6-butyl-12-formyl-5,6-dihydro-3,9-dimethoxyindolo[2,1-a]i-
soquinoline (5c) in 4 mL of EtOH. After addition of ca. 50 mg
of Na2CO3, the mixture was stirred overnight at room tem-
perature. Then most of the solvent was removed in vacuo
followed by addition of 30 mL of water. After extraction with
6-Bu t yl-12-for m yl-5,6-d ih yd r o-3,10-d ih yd r oxyin d olo-
[2,1-a ]isoqu in olin e (6d ): prepared in 65% yield from 6-butyl-
12-formyl-5,6-dihydro-3,10-dimethoxyindolo[2,1-a]isoquino-
line (5d )1 by a method similar to that described for 6a ;
crystallization from Et2O afforded beige crystals; dec >240 °C;

Inhibition of Tubulin Polymerization
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3531
3J BX ) 6 Hz, 1H, HB-5), 4.70 (m, 1H, H-6), 6.90-7.04 (m, 4H,
Et2O, the organic layer was washed with water and dried (Na2-
SO4). After evaporation of the solvent the product (96%) was
obtained as a crystalline solid. Recrystallization from MeOH/
Et2O (1:1) yielded colorless crystals: mp. 164 °C; 1H NMR
(CDCl3) δ 0.83 (t, J ) 7 Hz, 3H, -CH2-CH3), 1.18-1.62 (m, 6H,
3
Ar-H), 7.90 (s, 1H, OH), 8.11 (d, J ) 9 Hz, 1H, H-1), 8.18 (s,
1H, OH), 8.89 (s, 1H, OH); IR (KBr) 2206 cm-1
(C21H20N2O3) C, H, N.
. Anal.
6-Bu tyl-5,6-d ih yd r o-3,9-d im eth oxy-12-[(m eth ylim in o)-
m eth yl]in d olo[2,1-a ]isoqu in olin e (13a ). A 50-100-fold
excess of aqueous methylamine was added to a solution of 200
mg (0.55 mmol) of 6-butyl-12-formyl-5,6-dihydro-3,9-dimethox-
yindolo[2,1-a]isoquinoline (2c) in 10 mL of EtOH. After
addition of 0.5 mL of acetic acid, the mixture was stirred
overnight. After the removal of the solvent and the excess of
amine in vacuo, 50 mL of water was added. The aqueous
solution was extracted with Et2O. The combined organic
layers were washed twice with water and dried (Na2SO4). After
the evaporation of the solvent the product (92%) was dried in
vacuo. It was obtained as a brownish resin and required no
further purification: 1H NMR (CDCl3) δ 0.82 (t, J ) 7 Hz, 3H,
-CH2-CH3), 1.19-1.59 (m, 6H, -(CH2)3-CH3), 2.94 (dd, 2J AB/3J AX
2
-(CH2)3-CH3), 2.94 (dd, J AB/3J AX ) 15/1 Hz, 1H, -CH-H, HA-
2
5), 3.29 (dd, J AB/3J BX ) 15/6 Hz, 1H, -CH-H, HB-5), 3.87 (s,
3H, -OCH3), 3.90 (s, 3H, -OCH3), 4.53 (m, 1H, -CH-, H-6), 6.79-
6.96 (m, 5H, 4 × Ar-H, CdN-OH), 7.64 (d, J ) 9 Hz, 1H, Ar-
H), 8.07 (d, J ) 9 Hz, 1H, Ar-H), 8.74 (s, 1H, H-CdN-OH).
Anal. (C23H26N2O3) H, N; C: calcd, 72.99; found, 72.05.
6-Bu tyl-5,6-d ih yd r o-12-[(N-h yd r oxyim in o)m eth yl]-3,9,-
10-tr im eth oxyin d olo[2,1-a ]isoqu in olin e (10b): prepared in
96% yield from 5e by a method similar to that described for
the preparation of 10a ; purified by column chromatography
(SiO2; CH2Cl2/EtOAc, 7:1) and recrystallized from MeOH/Et2O
1
3
(1:1); mp 143-145 °C; H NMR (CDCl3) δ 0.83 (t, J ) 7 Hz,
3H, -CH2-CH3), 1.19-1.68 (m, 6H, -(CH2)3-CH3), 2.91, 2.97 (dd,
2J AB ) 15 Hz, 3J AX ) 1 Hz, 1H, HA-5), 3.27, 3.34 (dd, 2J AB ) 15
2
) 15/1 Hz, 1H, -CH-H, HA-5), 3.30 (dd, J AB/3J BX ) 15/6 Hz,
3
Hz, J BX ) 6 Hz, 1H, HB-5), 3.87 (s, 3H, -OCH3), 3.97 (s, 3H,
1H, -CH-H, HB-5), 3.59 (d, J ) 1 Hz, 3H, H-CdN-CH3), 3.88
(s, 3H, -OCH3), 3.89 (s, 3H, -OCH3), 4.53 (m, 1H, -CH-, H-6),
6.78-6.94 (m, 4H, Ar-H), 7.69 (d, J ) 9 Hz, 1H, Ar-H), 8.31
(d, J ) 9 Hz, 1H, Ar-H), 8.83 (d, J ) 1 Hz, 1H, H-CdN-CH3).
12-[(Be n zylim in o)m e t h yl]-6-b u t yl-5,6-d ih yd r o-3,9-
d im eth oxyin d olo[2,1-a ]isoqu in olin e (13b). A 50-100-fold
excess of benzylamine was added to a solution of 200 mg (0.55
mmol) of 6-butyl-12-formyl-5,6-dihydro-3,9-dimethoxyindolo-
[2,1-a]isoquinoline (2c) in 10 mL of EtOH. After addition of
ca. 100 mg of CaSO4 and 0.5 mL of acetic acid, the mixture
was stirred overnight. After the removal of the solvent and
the excess of amine in vacuo, 50 mL of water was added. The
aqueous solution was extracted with Et2O. The combined
organic layers were washed twice with water and dried (Na2-
SO4). After the evaporation of the solvent a solid product (80%)
was obtained which crystallized from EtOH to give colorless
-OCH3), 3.99 (s, 3H, -OCH3), 4.49-4.56 (m, 1H, H-6), 6.80 (s,
4
4
1H, H-8), 6.84 (d, J ) 2 Hz, 1H, H-4), 6.88, 6.91 (dd, J ) 2
Hz, 3J ) 9 Hz, 1H, H-2), 7.40-7.50 (br, 1H, OH), 7.62 (d, 3J )
9 Hz, 1H, H-1), 7.70 (s, 1H, H-11), 8.77 (s, 1H, H-CdN-OH).
Anal. (C24H28N2O4) C, H, N.
6-Bu tyl-12-cya n o-5,6-d ih yd r o-3,9-d im eth oxyin d olo[2,1-
a ]isoqu in olin e (11a ). Under nitrogen atmosphere, a solution
of 1 mL of Ac2O in 5 mL of dry pyridine was added dropwise
to a mixture of 190 mg (0.5 mmol) of 7 and 100 mg of
hydroxylamine hydrochloride in 5 mL of dry pyridine. The
reaction mixture was heated to 100 °C for 3 h. After cooling,
the mixture was poured into ice water and extracted with CH2-
Cl2. The combined organic layers were washed with 2 N HCl,
saturated NaHCO3 solution, and water and dried (Na2SO4).
After evaporation of the solvent and removal of the excess of
Ac2O in vacuo, the residue was chromatographed (SiO2; CH2-
Cl2/Et2O, 50:1). The crystalline product (91%) was recrystal-
lized from EtOH to give colorless crystals: mp 113-114 °C;
1H NMR (CDCl3) δ 0.8 (t, J ) 7 Hz, 3H, -CH2-CH3), 1.16-1.61
crystals: mp 101 °C; 1H NMR (CDCl3) δ (t, J ) 7 Hz, 3H,
2
-(CH2)3-CH3), 1.20-1.57 (m, 6H, -(CH2)3-CH3), 2.95 (dd, J AB
/
3J AX ) 15/1 Hz, 1H, -CH-H, HA-5), 3.30 (dd, J AB/3J BX ) 15/6
Hz, 1H, -CH-H, HB-5), 3.87 (s, 3H, -OCH3), 3.89 (s, 3H, -OCH3),
2
2
(m, 6H, -(CH2)3-CH3), 3.00 (dd, J AB/3J AX ) 16/1 Hz, 1H, -CH-
2
4.54 (m, 1H, -CH-, H-6), 4.86 (d, J AB ) 14 Hz, 1H, -CH-H-
H, HA-5), 3.37 (dd, 2J AB/3J BX ) 16/6 Hz, 1H, -CH-H, HB-5), 3.88
(s, 3H, -OCH3), 3.89 (s, 3H, -OCH3), 4.57 (m, 1H, -CH-, H-6),
6.81-6.83 (m, 2H, Ar-H), 6.89-6.96 (m, 2H, Ar-H), 7.60 (d, J
) 9 Hz, 1H, Ar-H), 8.31 (d, J ) 9 Hz, 1H, Ar-H); IR (KBr)
2210 cm-1. Anal. (C23H24N2O2) C, H, N.
2
C6H5), 4.95 (d, J AB ) 14 Hz, 1H, -CH-H-C6H5), 6.79 (d, J ) 2
Hz, 1H, Ar-H), 6.83-6.92 (m, 3H, Ar-H), 7.22-7.46 (m, 5H,
-C6H5), 7.67 (d, J ) 9 Hz, 1H, Ar-H), 8.43 (d, J ) 9 Hz, 1H,
Ar-H), 8.94 (s, 1H, H-CdN-); IR (KBr) 1620 cm-1
.
6-Bu tyl-5,6-d ih yd r o-3,9-d im eth oxy-12-[(m eth yla m in o)-
m eth yl]in d olo[2,1-a ]isoqu in olin e (14a ). Sodium borohy-
dride (50 mg) was added at 0 °C in small portions to a solution
of 0.5 mmol of 10a in 3 mL of MeOH. After stirring for 1 h at
room temperature the mixture was heated under reflux for
45 min. After evaporation of the solvent in vacuo, CH2Cl2 and
water (10 mL) were added and the layers separated. The
aqueous solution was extracted with CH2Cl2, and the combined
organic layers were washed with water and dried (Na2SO4).
After evaporation of the solvent, the crude product was purified
by chromatography over neutral alumina (activity III) with
CH2Cl2 and an increasing proportion of Et2O to give a light-
brown resin (60%): 1H NMR (CDCl3) δ 0.82 (t, J ) 7 Hz, 3H,
-CH2-CH3), 1.13-1.69 (m, 7H, -(CH2)3-CH3, NH), 2.59 (s, 3H,
6-Bu t yl-12-cya n o-5,6-d ih yd r o-3,9,10-t r im et h oxyin d o-
lo[2,1-a ]isoqu in olin e (11b): prepared in 77% yield from 10b
by a method similar to that described for 11a . The product
was purified by chromatography (SiO2; CH2Cl2/Et2O, 5:1) and
recrystallization from EtOH; mp 145-147 °C; 1H NMR (CDCl3)
δ 0.83 (t, 3J ) 7 Hz, 3H, -CH2-CH3), 1.15-1.61 (m, 6H, -(CH2)3-
2
3
CH3), 2.96, 3.02 (dd, J AB ) 16 Hz, J AX ) 1 Hz, 1H, HA-5),
2
3
3.35, 3.41 (dd, J AB ) 16 Hz, J BX ) 6 Hz, 1H, HB-5), 3.87 (s,
3H, -OCH3), 3.96 (s, 3H, -OCH3), 3.97 (s, 3H, -OCH3), 4.52-
4.60 (m, 1H, H-6), 6.80-6.82 (m, 2H, H-4, H-8), 6.90, 6.94 (dd,
3J ) 9 Hz, J ) 2 Hz, 1H, H-2), 7.14 (s, 1H, H-11), 8.27 (d, J
) 9 Hz, 1H, H-1); IR (KBr) 2199 cm-1. Anal. (C24H26N2O3) C,
H, N.
4
3
2
6-Bu tyl-12-cya n o-5,6-d ih yd r o-3,9-d ih yd r oxyin d olo[2,1-
a ]isoqu in olin e (12a ): prepared in 75% yield from 11a by a
method similar to that described for 6a ; after chromatography
(SiO2; CH2H2/Et2O, 2:1) beige crystals were obtained; mp 185-
186 °C; 1H NMR (DMSO-d6) 2-CH3), 1.15-1.43 (m, 6H, -(CH2)3-
-NH-CH3), 2.93 (dd, J AB/3J AX ) 15/1 Hz, 1H, -CH-H, HA-5),
2
3.28 (dd, J AB/3J BX ) 15/6 Hz, 1H, -CH-H, HB-5), 3.86 (s, 3H,
2
-OCH3), 3.89 (s, 3H, -OCH3), 4.00 (d, J AB ) 13 Hz, 1H, Ar-
2
CH-H-NH-), 4.15 (d, J AB ) 13 Hz, 1H, Ar-CH-H-NH-), 4.53
(m, 1H, -CH-, H-6), 6.73-6.84 (m, 3H, Ar-H), 6.91 (dd, J 1/2
)
2
CH3), 3.03 (dd, J AB/3J AX ) 16/1 Hz, 1H, -CH-H, HA-5), 3.27
9/3 Hz, 1H, Ar-H), 7.53 (d, J ) 9 Hz, 1H, Ar-H), 7.88 (d, J )
9 Hz, 1H, Ar-H); MS (EI) m/ z 378 (M•+). Anal. (C24H30N2O2)
C, H, N.
(dd, 2J AB/3J BX ) 16/6 Hz, 1H, -CH-H, HB-5), 4.73 (m, 1H, -CH-,
H-6), 6.76 (dd, J 1/2 ) 9/2 Hz, 1H, Ar-H), 6.84-6.87 (m, 2H,
Ar-H), 6.93 (d, J ) 2 Hz, 1H, Ar-H), 7.37 (d, J ) 9 Hz, 1H,
Ar-H), 7.99 (d, J ) 9 Hz, 1H, Ar-H), 9.50 (s, 1H, -OH), 10.06
(s, 1H, -OH); IR (KBr) 3290, 2210 cm-1. Anal. (C21H20N2O2)
H, N; C: calcd, 75.88; found, 75.40.
12-[(Be n zyla m in o)m e t h yl]-6-b u t yl-5,6-d ih yd r o-3,9-
d im eth oxyin d olo[2,1-a ]isoqu in olin e (14b): prepared from
10a in 73% yield by a method similar to that described for
1
11a to give a light-yellow resin; H NMR (CDCl3) δ 0.82 (t, J
6-Bu t yl-12-cya n o-5,6-d ih yd r o-3,9,10-t r ih yd r oxyin d o-
lo[2,1-a ]isoqu in olin e (12b): prepared in 78% yield from 11b
by a method similar to that described for 6e; gray crystals;
) 7 Hz, 3H, -CH2-CH3), 1.19-1.60 (m, 6H, -(CH2)3-CH3), 1.68
(s, br, 1H, NH), 2.92 (dd, 2J AB/3J AX ) 15/1 Hz, 1H, -CH-H, HA-
2
5), 3.26 (dd, J AB/3J BX ) 15/6 Hz, 1H, -CH-H, HB-5), 3.85 (s,
1
mp 200-202 °C; H NMR (CDCl3) δ 0.81 (t, 3J ) 7 Hz, 3H,
3H, -OCH3), 3.88 (s, 3H, -OCH3), 3.98 (d, J ) 4 Hz, 2H, -NH-
CH2-C6H5), 4.01 (d, 2J AB ) 13 Hz, 1H, Ar-CH-H-NH-), 4.19 (d,
2J AB ) 13 Hz, 1H, Ar-CH-H-NH-), 4.51 (m, 1H, -CH-, H-6),
-CH2-CH3), 1.21-1.58 (m, 6H, -(CH2)3-CH3), 3.07, 3.13 (dd, 2J AB
3
2
) 16 Hz, J AX ) 1 Hz, 1H, HA-5), 3.33, 3.39 (dd, J AB ) 16 Hz,

3532 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Goldbrunner et al.
6.74-6.84 (m, 4H, Ar-H), 7.28-7.47 (m, 6H, -C6H5, Ar-H), 7.79
(d, J ) 9 Hz, 1H, Ar-H). Anal. (C30H34N2O2) C, H, N.
Sep a r a tion of th e En a n tiom er s of 6b,c. The enanti-
omers of 6b,c were separated semipreparatively by liquid
chromatography on triacetylcellulose (20-30 µm)30 with EtOH
as eluent. The purity of the separated enantiomers was
determined by HPLC (56 bar, 30 mL/min for 6b; 26 bar, 15
mL/min for 6c) using tris(phenylcarbamoyl)cellulose on SiO2
as stationary phase, EtOH as eluent, and both UV spectro-
scopic (278 nm) and polarimetric (436 nm) detection. Capacity
factors and optical purities: (+)-6b, k′(+)436 ) 0.04, P ) 0.99;
(-)-6b, k′(-)436 ) 0.16, P ) 0.94; (+)-6c, k′(+)436 ) 0.01, P )
1.00; (-)-6c, k′(-)436 ) 0.13, P ) 0.97.
Ma ter ia ls a n d Rea gen ts for Bioa ssa ys. Drugs and
biochemicals were obtained from Sigma (Deisenhofen, Ger-
many). [3H]Colchicine was purchased from New England
Nuclear (Dreieich, Germany). Buffer solutions used: PEMs0.1
M PIPES-NaOH, 1 mM EGTA, 1 mM MgSO4, pH 6.6-6.7;
PEMGs0.1 M PIPES-NaOH, 0.8 M monosodium L-glutamate,
1 mM EGTA, 1 mM MgSO4, pH 6.6-6.7; PBSs8.0 g/L NaCl,
0.2 g/L KCl, 1.0 g/L Na2HPO4‚H2O, 0.2 g/L KH2PO4. Scintil-
lation liquid: Rotiszint eco plus (Roth, Karlsruhe, Germany).
Isola tion a n d P u r ifica tion of Ca lf Br a in Tu bu lin . The
cortex of one or two fresh calf brains in ice-cold PEM buffer (1
mL/g of tissue, + 16 mg of DTE/100 mL of buffer solution)
was homogenized in portions. After centrifugation (90 min,
20000g) at 2-4 °C, the supernatant was carefully decanted.
The concentrations of GTP and ATP were adjusted to 0.1 and
2.5 mM, respectively. After stirring gently at 37 °C for 30 min
the solution was transferred to centrifugation tubes and
carefully underlayered with a prewarmed (37 °C) sucrose
solution (10% in PEM buffer solution containing 1 mM GTP,
ca. 10% of the transferred volume). After centrifugation at
37 °C for 45 min (20000g) the pellets were weighed, suspended
in ice-cold PEM buffer solution (3 mL/g), and homogenized in
a Teflon-in-glas potter. After standing in ice for 30 min, the
suspension was centrifuged at 2 °C for 30 min (40000g). The
supernatant was removed and adjusted to 1 mM GTP. By
incubation at 37 °C for 15 min tubulin was polymerized once
again. After centrifugation at 37 °C for 30 min microtubuli
were obtained as a shiny gel-like pellet. The yields ranged
from 2 to 6 g/brain. Aliquots were frozen in liquid nitrogen
and stored at -70 °C. Purity was checked by polyacrylamide
gel electrophoresis.
mixture (0.1 mL) was filtered under reduced vacuum through
a stack of four DEAE-cellulose paper filters and washed four
times with 10 mL of PEM buffer. Radioactivity adsorbed on
the filter representing tubulin-bound [3H]colchicine was quan-
tified in a liquid scintillation counter. Reaction mixtures
without tubulin gave the background values.
Deter m in a tion of Cytosta tic Activity: 1. MDA-MB 231
Hu m a n Br ea st Ca n cer Cells. Hormone-independent human
MDA-MB 231 breast cancer cells were obtained from American
Type Culture Collection (ATCC, Rockville, MD). Cells were
grown in McCoy-5a medium, supplemented with L-glutamine
(73 mg/L), gentamycin sulfate (50 mg/L), NaHCO3 (2.2 g/L),
and 5% sterilized fetal calf serum (FCS). At the start of the
experiment, the cell suspension was tranferred to 96-well
microplates (100 µL/well). After the cells grew for 2-3 days
in a humidified incubator with 5% CO2 at 37 °C, medium was
replaced by one containing the test compounds (200 µL/well).
Control wells (16/plate) contained 0.1% DMF that was used
for the preparation of stock solutions. Initial cell density was
determined by addition of glutaric dialdehyde (1% in PBS; 100
µL/well) instead of test compound. After incubation for about
4 days, medium was removed and 100 µL of glutaric dialde-
hyde in PBS (1%) was added for fixation. After 15 min, the
solution of aldehyde was decanted. Cells were stained by
treating them for 25 min with 100 µL of an aqueous solution
of crystal violet (0.02%). After decanting, cells were washed
several times with water to remove adherent dye. After
addition of 100 µL of EtOH (70%), plates were gently shaken
for 2 h. Optical density of each well was measured in a
microplate autoreader EL 309 (Bio-tek) at 578 nm.
2. MCF -7 Hu m a n Br ea st Ca n cer Cells. A similar
procedure to that described for MDA-MB 231 cells was applied
with alterations: Cells were grown in EMEM supplemented
with sodium pyruvate (110 mg/L), gentamycin sulfate (50 mg/
L), NaHCO3 (2.2 g/L), and 10% FCS. Medium that contained
test compounds was supplemented with dextran-charcoal
(DCC)-treated FCS to avoid interference with steroidal hor-
mones in the serum. Incubation with inhibitor lasted ca. 8
days.
Ack n ow led gm en t. We wish to thank Renate Liebl
for excellent technical assistance and Robert Gastpar
for helpful discussions.
Tu bu lin P olym er iza tion Assa y. The pellet of frozen
microtubuli was warmed to 37 °C in a water bath. After
addition of the 20-fold volume of ice-cold PEMG buffer, it was
homogenized. Depolymerization was completed by keeping the
mixture at 0 °C for 30 min followed by centrifugation at 2 °C
(30 min, 30000g) to remove insoluble protein. Each reaction
tube contained 0.46 mL of the supernatant and 20 µL of the
DMSO solution of the test compound in varying concentra-
tions. Reaction mixtures were preincubated at 37 °C for 15
min and chilled on ice followed by addition of 20 µL of a 25
mM GTP solution in PEMG buffer to each tube. Reaction
mixtures were transferred to cuvettes of a UV spectrophotom-
eter connected to two different temperature controllers. First,
the temperature inside the cuvettes was held at 2 °C. The
cuvette holder was then switched to the second temperature
contoller at 37 °C, and the absorption was measured over a
period of 20 min at 350 nm. Absorption at the start of the
reaction was used as baseline. Two independent experiments
were performed for the standard concentration (40 µM) and
the three for determination of IC50 values. Each experiment
had two control reaction mixtures; their mean value was
defined as 100% and their turbidity readings were generally
within 10% of each other.
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Inhibition of Tubulin Polymerization
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