Methoxy-substituted 3-formyl-2-phenylindoles inhibit tubulin polymerization

Article abstract of DOI:10.1021/jm980228l

The aim of this study was the identification of the essential structural elements in the 12-formyl-5,6-dihydroindolo[2,1-a]isoquinoline system required for the inhibition of tubulin polymerization which is understood to be the predominant mode of action o

Full text of DOI:10.1021/jm980228l

J . Med. Chem. 1998, 41, 4965-4972
4965
Meth oxy-Su bstitu ted 3-F or m yl-2-p h en ylin d oles In h ibit Tu bu lin P olym er iza tion
Robert Gastpar,† Michael Goldbrunner,† Doris Marko,‡ and Erwin von Angerer*,†
Institut fu¨r Pharmazie, Universita¨t Regensburg, D-93040 Regensburg, Germany, and Fachbereich Chemie,
Fachrichtung Lebensmittelchemie und Umwelttoxikologie, Universita¨t Kaiserslautern, D-67663 Kaiserslautern, Germany
Received April 13, 1998
The aim of this study was the identification of the essential structural elements in the 12-
formyl-5,6-dihydroindolo[2,1-a]isoquinoline system required for the inhibition of tubulin
polymerization which is understood to be the predominant mode of action of this class of
cytostatics. Since 2-phenylindole forms the main fragment of this tetracycle, it was used as
the basic structure and modified with respect to the number and positions of the oxygen
functions in the aromatic rings. Further modifications related to the nitrogen, which was both
replaced by oxygen and sulfur and alkylated. All derivatives were tested for cytostatic activity
in human breast cancer cells (MDA-MB 231, MCF-7) and inhibition of tubulin polymerization.
The spectrum of activity ranged from inactive to IC50 values of 35 nM (cell growth inhibition)
and 1.5 µM (tubulin polymerization), respectively, for the most active derivative 3e (3-formyl-
6-methoxy-2-(4-methoxyphenyl)indole). Although the correlation between antiproliferative
activity and inhibition of tubulin polymerization was not very pronounced, all of the potent
cytostatic agents in this study disrupted microtubule assembly completely at the standard
concentration of 40 µM. By fluorescence microscopy it was demonstrated that the derivative
3e degrades the cytoskeleton in a similar fashion as colchicine does leading to the condensation
of the microtubules around the nucleus after treatment. The comparison between hydroxy
and methoxy derivatives revealed a striking difference between the 2-phenylindole derivatives
and the indoloisoquinolines. In the 2-phenylindole series, the methoxy compounds were much
more effective than the free phenols, whereas in the tetracyclic system the effect of the hydroxy
derivatives exceeded that of the methylated compounds by 1 order of magnitude. Preliminary
studies on the binding mode showed that both the 2-phenylindole derivatives and the
indoloisoquinolines bind to the colchicine site on tubulin.
The discovery of a variety of natural products that
interact with the tubulin system either by preventing
the polymerization of the R-/â-tubulin heterodimers to
microtubules or by stabilizing the polymeric structures
greatly stimulated the interest in this cellular system
as a target for anticancer drugs. The assembly of
microtubules is understood to be a dynamic process of
polymerization and depolymerization. One of the im-
portant functions of the microtubules is the formation
of the mitotic spindle necessary for the correct distribu-
tion of the chromosomes in dividing cells. Therefore,
compounds acting on this system have become a valu-
able alternative to drugs that interact with the DNA
as has been demonstrated by the therapeutic use of the
vinca alkaloids vincristine and vinblastine. These
alkaloids and some other substances of natural origin
bind to the dimeric form of tubulin at distinct sites and
prevent the formation of functional microtubules.1
Since the function of the microtubules depends on the
dynamic equilibrium between dimeric and polymeric
structures, the stabilization of the microtubules also
inhibits cell growth as had been demonstrated for
paclitaxel (Taxol). After the discovery of this drug a
variety of other natural products with structures of
similar complexity and high biological activity were
identified including epothilone B,2,3 discodermolide,4-6
and eleutherobin.7
From a synthetic point of view, compounds of low
molecular weight are more attractive as a lead. The
chemical structure of colchicine,8 the first antimitotic
agent identified, gave hints toward important structural
elements required for the interference with the micro-
tubule assembly. Some of these elements were found
in podophyllotoxin9 and combretastatin A-4,10 two other
inhibitors of tubulin polymerization. Over the past few
years the spectrum of compounds which inhibit the
growth of tumor cells by disrupting the dynamic equi-
librium between the dimers and polymeric structures
has steadily expanded. Among these substances are
synthetic analogues of colchicine11,12 and combretastatin
A,13,14 various heterocyclic systems such as 2-phenyl-
quinolinone,15 2-styrylquinazolin-4-one,16 2-phenyl-1,8-
naphthyridin-4-ones,17,18 steroidal structures derived
from 2-methoxyestradiol,19 2,3-diaryl-1,1-dichlorocyclo-
propanes,20 and the sulfonamide E 701021 (Chart 1). The
experimental data accumulated over the past decade
have recently been analyzed by advanced quantitative
structure-activity relationship (QSAR) techniques.22
In a recent paper we showed that the cytostatic effect
of hydroxylated 12-formyl-5,6-dihydroindolo[2,1-a]iso-
quinolines can be attributed to their inhibitory effects
on tubulin polymerization.23 The aim of the present
study was the identification of the essential structural
elements in this class of heterocycles required for the
disruption of microtubule assembly. Since the in-
dolo[2,1-a]isoquinoline skeleton can be considered as a
† Universita¨t Regensburg.
‡ Universita¨t Kaiserslautern.
10.1021/jm980228l CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/13/1998

4966 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Ch a r t 1. Inhibitors of Tubulin Polymerization
Gastpar et al.
nitrogen which was both alkylated and replaced by
oxygen and sulfur.
Ch em istr y
As a general approach to the synthesis of the 2-phen-
ylindole system, the Bischler method was applied
(Scheme 1). Ring-substituted aniline derivatives were
reacted with ω-bromoacetophenone and its 4-methoxy
congener to give the corresponding 2-phenylindole 1.24
N-Alkylation was readily achieved by deprotonation of
1 with sodium hydride followed by the reaction with the
appropriate alkyl halide.25 The preparation of the
analogous 2-phenylbenzo[b]furan26 and 2-phenylben-
zo[b]thiophene27 has been described previously. The
formyl group was introduced into the 3-position of the
heterocycles by the Vilsmeier reaction using dimethyl-
formamide and POCl3. In some cases, the ether func-
tions were cleaved with boron tribromide to afford the
phenolic compounds 4.
Biologica l Resu lts a n d Discu ssion
Antiproliferative activity was determined in two hu-
man breast cancer cell lines, MDA-MB 231 and MCF-
7, that are often used for the evaluation of antimitotic
compounds.20,28-30 The latter cell line is estrogen recep-
tor-positive and responds to estrogens and antiestro-
gens. Since hydroxylated 2-phenylindoles24 and ana-
logues27 have been shown to exert their cytostatic action
via the estrogen receptor, MCF-7 cells were included in
order to detect potential (anti)hormonal effects.
Preliminary experiments showed that contrary to the
5,6-dihydroindolo[2,1-a]isoquinoline series the methoxy
derivatives of 3-formyl-2-phenylindole were more active
than the corresponding hydroxy compounds. For the
identification of the structural requirements for cyto-
static activity, one or both methoxy groups were omitted.
Cytostatic activity decreased only slightly upon this
modification (Table 1). Stronger effects were noted
when the phenyl ring (3-formylindole) or the indole
nitrogen (5) was removed or the NH group had been
bridged 2-phenylindole, a number of 3-formyl-2-phen-
ylindoles with oxygen functions at one or both aromatic
rings were synthesized and evaluated for inhibitory
activity on tubulin polymerization and cell proliferation.
Additional structural variations concerned the indole
Sch em e 1a
a
(A) N,N-Dimethylaniline/170 °C; (B) NaH/R3-Br; (C) DMF/POCl3; (D) BBr3.

Methoxy-Substituted 3-Formyl-2-phenylindoles
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 4967
Ta ble 1. Inhibitory Effect of 3-Formyl-2-phenylindoles and Analogues on the Growth of Breast Cancer Cells and Tubulin
Polymerization
inhibn of tubulin
inhibn of cell growth
polymerization, %c
d
compd
R1
R2
X
MDAa
MCF-7b
(IC50
2 ( 7
1 ( 6
)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
4a
4b
4c
H
H
H
NH
0.42 ( 0.07
0.24 ( 0.02
0.47 ( 0.02
0.26 ( 0.03
0.035 ( 0.004
0.65 ( 0.08
0.19 ( 0.08
0.18 ( 0.04
0.18 ( 0.07
0.16 ( 0.03
6.6 ( 0.3
6-OCH3
H
NH
NH
NH
NH
NH
NH
NH
NH
NCH3
NCH3
NC5H11
O
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
97 ( 4 (3.3 µM)
93 ( 4 (4.0 µM)
98 ( 3 (1.5 µM)
14 ( 5
5-OCH3
6-OCH3
4,7-(OCH3)2
5,6-(OCH3)2
5,7-(OCH3)2
6-F
5-OCH3
6-OCH3
6-OCH3
5-OCH3
6-OCH3
5-OH
>10
0.22 ( 0.05
2.8 ( 0.2
0.049 ( 0.006
0.23 ( 0.01
1.7 ( 0.1
5 ( 6
48 ( 8
0.043 ( 0.009
92 ( 5 (1.8 µM)
4 ( 4
>10
>10
2.4 ( 0.2
3.3 ( 0.2
8.4 ( 1.6
0.99 ( 0.12
2.5 ( 0.4
7.9 ( 0.4
9.3 ( 0.2
5.4 ( 0.1
2.4 ( 0.2
48 ( 3
4 ( 1
>10
35 ( 9
S
0.23 ( 0.03
6.7 ( 0.2
3.3 ( 0.1
100 ( 3 (1.8 µM)
28 ( 9
NH
NH
S
6-OH
6-OH
H
OH
8 ( 4
>10
>10
21 ( 7
3-formylindole
5
colchicine
>10
0 ( 5
7.9 ( 0.4
0.03 ( 0.01
7.0 ( 0.1
11 ( 7
nde
nd
100 ( 5 (1.9 µM)
7a f
OCH3
OCH3
OH
C3H7
C4H9
C3H7
C4H9
(NR)
(NR)
(NR)
(NR)
>10
17
11
7bf
8.7
0.3
1.4
nd
0.29
0.22
7cf
93 (19 µM)
100 (8.9 µM)
7d f
OH
a
Inhibition of the growth of MDA-MB 231 human breast cancer cells after incubation for 4 days, determined by measuring optical
b
densities after crystal violet staining of vital cells. Mean of eight replicates ( SD. Inhibition of the growth of estrogen-sensitive MCF-7
human breast cancer cells after incubation for 4 days, determined by measuring optical densities after crystal violet staining of vital
cells. Mean of eight replicates ( SD. c Inhibition of tubulin polymerization by a 40 µM solution of test compounds, measured after 20 min
d
at 37 °C. Mean values of two independent experiments. Full details are given in the text. IC50 values were determined for all compounds
that showed inhibition >50% at 40 µM. e nd, not determined. Data from ref 23.
f
replaced by oxygen (3m ). N-Alkyation also diminished
the antiproliferative effect, whereas replacement of the
indole nitrogen by sulfur (3n ) had only a minor effect.
The most favorable conditions were provided by a
methoxy group (3e) or a fluorine atom (3i) in position 6
of the indole with IC50 values of 0.035 µM (MCF-7: 0.16
µM) and 0.049 µM (MCF-7: 0.043 µM), respectively.
Additional methoxy groups did not improve the anti-
proliferative effect. The equivalence of fluoro and
methoxy substituents was also noticed in the 2-phen-
ylquinolone series.31 The comparison of the data from
the estrogen receptor-positive (MCF-7) and -negative
(MDA-MB 231) cell lines revealed no significant differ-
ences of the IC50 values. This result was not unexpected
because most of these compounds do not meet the
structural requirements for high binding affinity for the
estrogen receptor such as free hydroxy groups and
lipophilic substituents in the central part of the mol-
ecule.1 Since both elements interfere with the tubulin-
directed action (vide infra), no attempt was made to
design molecules that act via both the tubulin system
and the estrogen receptor.
tion inhibition ranged from 0% to 100% depending on
the molecular structure. The correlation between these
data and those from the antiproliferative assays was
rather poor. However, all of compounds which com-
pletely suppressed microtubule assembly at this con-
centration also strongly inhibited cellular growth. Simi-
lar deviations between these two different biological
assays were also observed in other classes of antimitotic
agents, e.g., in the diaryldichlorocyclopropane series20
and the curacin A analogues.32
The most potent derivatives (3c-e,i) were tested at
various concentrations. The dose-response curves
depicted in Figure 1 displayed inhibitory activities
similar to that of colchicine. The IC50 values were
approximately 1 order of magnitude lower than those
of the most potent indoloisoquinolines (Table 1). The
main difference between the 2-phenylindoles and
isosters and the indoloisoquinoline system is the dis-
crepancy between the hydroxy and methoxy forms as
the more active structures. This finding can possibly
be rationalized by different binding modes or binding
sites of these compounds. Since the indolo[2,1-a]iso-
quinolines were shown to bind at the colchicine binding
site,23 two different 3-formyl-2-indoles (3e,k ) and the
thiophene analogue of 3e (3n ), as well as two represen-
tative examples of the indoloisoquinoline series (7a ,c),
were tested for their capability of competing with
All of the compounds were tested for their ability to
interfere with the tubulin polymerization. In the rou-
tine experiments a standard dose of 40 µM was used
and the degree of polymerization was determined tur-
bidimetrically at 350 nm (Table 1). At this concentra-

4968 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Gastpar et al.
the 2-phenylindole series, the inhibitory effect of the
methoxy compounds exceeded that of the phenols by 1
order of magnitude, whereas the situation in the tetra-
cyclic system was exactly reversed. From the data
available it appears that the formyl group and the
aromatic rings are the most important structural ele-
ments for the interaction with tubulin. Despite the fact
that the active forms of the two classes of inhibitors
differ in the nature of the aromatic oxygen functions,
they are both capable of displacing cochicine from its
binding site. Obviously, the heterocyclic 3-formyl group
is the dominant structural element, and the oxygen
functions can possibly bind to different sites depending
on their chemical structure.
The formyl group can be considered as a novel
structural element in this class of antimitotic agents.
There is only a vague analogy to the 2-phenylquinolone
system which contains a six-membered heterocycle with
an incorporated carbonyl function (Chart 1). When the
latter system was substituted with methoxy or fluorine,
its activity on tubulin polymerization and cellular
growth was rather similar to that of the 2-phenylindole
structure.31 The strong inhibitory effects of some of
these 2-phenylindole derivatives on tumor cell growth
and tubulin polymerization make this system attractive
for further structural variations which might increase
the cytostatic potency of this heterocyclic structure.
F igu r e 1. Inhibitory effect of various 3-formyl-2-phenylindoles
and of colchicine on the polymerization of calf brain tubulin.
Assembly of microtubules was assessed turbidimetrically at
350 nm 20 min after the temperature had been switched from
2 to 37 °C. Polymerization in the absence of inhibitor gave the
control readings. Values are means of three independent
experiments ( SEM.
Ta ble 2. Inhibition of Colchicine Binding by
3-Formyl-2-phenylindoles and Analogues
inhibn of colchicine
compd
bindinga (%)
Exp er im en ta l Section
3e
3k
3n
7a
7c
23
30
56
<10
40
Melting points were determined on a Bu¨chi 510 apparatus
and are uncorrected. Elemental analyses were performed by
the Mikroanalytisches Laboratorium, University of Regens-
burg, and were within (0.40% of the calculated values except
where noted. IR spectra were recorded on an Acculab 7
infrared spectrometer (Beckman), and peak positions are
expressed in cm-1. NMR spectra were obtained on a Bruker
AC-250 spectrometer with TMS as internal standard. They
were recorded in DMSO-d6 as solvent except where noted and
are consistent with the assigned structures; values are given
in δ units (ppm). The syntheses of the 2-phenylindoles 1a ,36
1b,37 1c,38 1d ,24 1e,24 1f,39 2a ,24 and 2d ,24 5-methoxy-2-(4-
methoxyphenyl)benzo[b]furan,40 6-methoxy-2-(4-methoxyphen-
yl)benzo[b]thiophene,41 and (Z)-2,3-bis(4-methoxyphenyl)-
propenal (5)42 have been described previously.
Gen er a l P r oced u r e for th e Syn th esis of 2-P h en ylin -
d oles. The respective aniline (60 mmol) was dissolved in 15
mL of N,N-dimethylaniline and heated to 170 °C. A solution
of 20 mmol of the ω-bromoacetophenone in 30 mL of xylene
was added dropwise. After addition, the reaction mixture was
heated under reflux for 3 h. After the mixture had cooled,
150 mL of EtOAc and 70 mL of 2 N HCl were added and the
layers separated. The aqueous layer was extracted with
EtOAc several times. The combined organic layers were
washed with water and saline and dried (MgSO4). After
evaporation of the solvents the crude product was purified by
chromatography (SiO2) and/or crystallization. The following
compounds were obtained by this method.
Reaction mixtures contained tubulin, 1 µM [3H]colchicine, and
a
10 µM inhibitor and were incubated for 30 min at 37 °C prior to
analysis. Mean of two independent experiments.
colchicine for binding to tubulin. In preliminary experi-
ments with a standard concentration of 10 µM, we found
that all of these agents are capable of displacing
[3H]colchicine from its binding site (Table 2). Since none
of the compounds stabilized the colchicine binding as
the vinca site ligands do,21,23,33-35 an interaction with
the binding of vinblastine is not very likely.
The degradation of the cytoskeleton as the result of
the interference with the microtubule assembly can be
demonstrated by fluorescence microscopy. Representa-
tive cells treated with 3e and colchicine in concentra-
tions of 0.1, 1, and 10 µmol/L are shown in Figure 2.
Microtubules were visualized by the double-antibody
technique with Cy3 as the fluorescence label. Both
indole 3e and colchicine distroyed the cellular network
of the microtubules at 0.1 µM and led to a marked
tubulin condensation around the nucleus when higher
concentrations were applied.
The results from the various assays suggest that the
antitumor activity of the 12-formyl-5,6-dihydroindolo[2,1-
a]isoquinolines is associated with the 2-phenylindole
structure incorporated in the tetracyclic system. The
removal of the alkane bridge that links carbon 3 of the
indole with carbon 2 of the 2-phenyl moiety increased
the potency substantially. In both series, 2-phenyl-
indoles and indoloisoquinolines, methoxy and hydroxy
derivatives were tested. The results revealed a striking
difference between these two classes of compounds. In
5,6-Dim et h oxy-2-(4-m et h oxyp h en yl)in d ole (1g): yield
16%; colorless crystals; mp 207-209 °C (EtOH); 1H NMR 3.72
(s, 3H, -OCH3), 3.77 (s, 6H, -OCH3), 6.50-7.00 (m, 1H, Ar-
H), 6.54 (d, 4J ) 2 Hz, 1H, H-3), 6.93/7.63 (AA′BB′, 3J ) 9 Hz,
4H, Ar-H), 6.97 (s, 2H, H-4, H-7). Anal. (C17H17NO3) C, H,
N.
5,7-Dim eth oxy-2-(4-m eth oxyp h en yl)in d ole (1h ): yield
19%; colorless crystals; mp 164-166 °C (EtOH); 1H NMR 3.72
(s, 3H, -OCH3), 3.80 (s, 3H, -OCH3), 3.89 (s, 3H, -OCH3),
4
6.20-7.80 (m, 1H, Ar-H), 6.23 (d, J ) 2 Hz, 1H, H-6), 6.50
4
(d, J ) 2 Hz, 1H, H-4), 6.56 (s, 1H, H-3), 6.89/7.77 (AA′BB′,
3J ) 9 Hz, 4H, Ar-H). Anal. (C17H17NO3) C, H, N.

Methoxy-Substituted 3-Formyl-2-phenylindoles
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 4969
F igu r e 2. Effect of 3-formyl-6-methoxy-2-(4-methoxyphenyl)indole (3e) and colchicine on the cellular distribution of tubulin,
determined by fluorescence microscopy. MCF-7 cells were grown on glass slides overnight at 37 °C and subsequently treated with
the drugs at various concentrations for 60 min. After fixation, microtubules were visualized by sequential treatment with a murine
monoclonal antibody to R-tubulin and a Cy3-labeled goat antibody to mouse IgG: (A) untreated cells; (B) cells treated with 3e,
0.1 µM; (C) cells treated with 3e, 1 µM; (D) cells treated with 3e, 10 µM; (E) cells treated with colchicine, 0.1 µM; (F) cells treated
with colchicine, 1 µM; (G) cells treated with colchicine, 10 µM. For the photographs presented, the magnification was 1200-fold
(figure reproduced at 65% of original).
6-F lu or o-2-(4-m eth oxyp h en yl)in d ole (1i): yield 20%;
at a temperature of 15 °C. After the mixture stirred for 10
min, 2.0 mmol of the respective aromatic compound in 10 mL
of dry DMF was added to the viscous mixture while the
temperature was kept between 10 and 20 °C. The reaction
was completed by stirring for 1 h at 40 °C. The mixture was
poured into 100 mL of ice-water. The pH was adjusted to 6
by adding NaOH solution (40%). After extraction with CHCl3,
the combined organic layers were washed with water and
saline and dried (MgSO4). After evaporation of the solvent,
the residue was purified by chromatography (SiO2) and/or
crystallization. The following compounds were synthesized by
this method.
1
white crystals; mp 208-210 °C (EtOH); H NMR 3.92 (s, 3H,
3
-OCH3), 6.74-7.99 (m, 5H, Ar-H), 7.13/7.88 (AA′BB′, J ) 9
Hz, 4H, Ar-H). Anal. (C15H12FNO) C, H, N.
6-Meth oxy-2-(4-m eth oxyp h en yl)-1-p en tylin d ole (2e).
Under N2, a solution of 1e (4.3 mmol) in 25 mL of dry DMF
was added slowly to an ice-cold suspension of sodium hydride
(6.0 mmol) in 10 mL of dry DMF. Stirring was continued until
the gas evolution ceased. 1-Bromopentane (4.3 mmol) in 30
mL of dry DMF was added dropwise. After the mixture stirred
for 2 h at room temperature, the excess of sodium hydride was
carefully destroyed by addition of water. After addition of an
additional volume of 80 mL of water, the mixture was
extracted with EtOAc several times. The combined organic
layers were washed with water and dried (MgSO4). After
evaporation of the solvent, the product was purified by
chromatography (SiO2, CH2Cl2) and crystallized from EtOH
to afford colorless crystals (62%): mp 62-64 °C; 1H NMR
0.58-1.71 (m, 9H, alkyl-H), 3.81 (s, 6H, -OCH3), 4.31 (t, 3J )
7 Hz, 2H, N-CH2-), 6.30-7.43 (m, 2H, Ar-H), 6.30 (s, 1H,
H-7), 6.67 (dd, 3J ) 9 Hz, 4J ) 2 Hz, 1H, H-5), 7.00/7.37
3-F or m yl-2-p h en ylin d ole (3a ): 31% yield; light-brown
1
powder; mp 236-240 °C; H NMR 7.09-7.93 (m, 9H, Ar-H),
8.08-8.33 (m, 1H, H-4), 9.97 (s, 1H, formyl-H). Anal.
(C15H11NO) C, H, N.
3-F or m yl-6-m eth oxy-2-p h en ylin d ole (3b): 23% yield;
yellow crystals; mp 255-258 °C; 1H NMR 3.78 (s, 3H, -OCH3),
3
6.82 (d, J ) 9 Hz, 1H, H-5), 6.90 (s, 1H, H-7), 7.34-7.78 (m,
3
6H, Ar-H), 7.98 (d, J ) 9 Hz, 1H, H-4), 9.78 (s, 1H, formyl-
H). Anal. (C16H13NO2) C, H, N.
3
(AA′BB′, J ) 9 Hz, 4H, Ar-H). Anal. (C21H25NO2) C, H, N.
3-F or m yl-2-(4-m eth oxyp h en yl)in d ole (3c): 20% yield;
Gen er a l P r oced u r e for th e F or m yla tion Rea ction .
white crystals; mp 206-208 °C; 1H NMR 3.88 (s, 3H, -OCH3),
3
Under N2, POCl3 (20 mmol) was added to 15 mmol of dry DMF
7.10-7.92 (m, 4H, Ar-H), 7.19/7.75 (AA′BB′, J ) 9 Hz, 4H,

4970 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Gastpar et al.
3
3
Ar-H), 8.12-8.36 (m, 1H, H-4), 10.03 (s, 1H, formyl-H). Anal.
(C16H13NO2) C, H, N.
7.11/7.54 (AA′BB′, J ) 9 Hz, 4H, Ar-H), 8.07 (d, J ) 9 Hz,
1H, H-4), 9.53 (s, 1H, formyl-H). Anal. (C22H25NO3) C, H, N.
3-F or m yl-5-m eth oxy-2-(4-m eth oxyp h en yl)in d ole (3d ):
yield 46%; light-pink crystals; mp 230-232 °C (MeOH); IR
(KBr) 1630; 1H NMR 3.79 (s, 3H, -OCH3), 3.87 (s, 3H,
3-F or m yl-5-m eth oxy-2-(4-m eth oxyp h en yl)ben zo[b]fu -
r a n (3m ): 64% yield; white crystals; mp 122-124 °C (EtOH);
1H NMR 3.82 (s, 3H, -OCH3), 3.86 (s, 3H, -OCH3), 6.99 (dd,
3
4
4
3
3J ) 9 Hz, J ) 2 Hz, 1H, H-6), 7.17/7.93 (AA′BB′, J ) 9 Hz,
-OCH3), 6.70-7.75 (m, 1H, Ar-H), 6.78 (dd, J ) 9 Hz, J )
2 Hz, 1H, H-6), 7.13/7.69 (AA′BB′, 3J ) 9 Hz, 4H, Ar-H), 7.37
3
4
4H, Ar-H), 7.58 (d, J ) 9 Hz, 1H, H-7), 7.60 (d, J ) 2 Hz,
1H, H-4), 9.28 (s, 1H, formyl-H). Anal. (C17H14O4) C, H.
3-For m yl-6-m eth oxy-2-(4-m eth oxyph en yl)ben zo[b]th io-
p h en e (3n ): 58% yield; light-yellow powder; mp 122-124 °C;
IR (KBr) 1680; 1H NMR (CDCl3) 3.71 (s, 3H, -OCH3), 3.81 (s,
3H, -OCH3), 6.75-7.43 (m, 2H, Ar-H), 6.84/7.34 (AA′BB′, 3J
3
4
(d, J ) 9 Hz, 1H, H-7), 7.70 (d, J ) 2 Hz, 1H, H-4), 10.09 (s,
1H, formyl-H); 13C NMR (DMSO) 55.40 (2C), 103.05, 112.56,
113.00, 113.12, 114.47 (2C), 122.22, 126.79, 130.67, 131.07
(2C), 149.16, 155.82, 160.53, 185.18. Anal. (C17H15NO3) C, H,
N.
3
3-F or m yl-6-m eth oxy-2-(4-m eth oxyp h en yl)in d ole (3e):
) 9 Hz, 4H, Ar-H), 8.50 (d, J ) 9 Hz, 1H, H-4), 9.88 (s, 1H,
1
formyl-H); 13C NMR (CDCl3) 55.47 (2C), 104.54, 114.43 (2C),
115.50, 124.10, 125.77, 129.53, 131,18, 131.79 (2C), 139.24,
158.26, 158.54, 161.13, 186.61. Anal. (C17H14O3S) C, H.
3-F or m yl-5-h yd r oxy-2-(4-h yd r oxyp h en yl)in d ole (4a ).
Under N2, a suspension of 3-formyl-5-methoxy-2-(4-methoxy-
phenyl)indole (3d ) (1.0 mmol) in 150 mL of dry CH2Cl2 was
added slowly to a solution of 1.0 mL (10.8 mmol) of BBr3 in 10
mL of dry CH2Cl2 at a temperature of -10 °C. After stirring
for 1 h at this temperature, the mixture was allowed to warm
to room temperature, and stirring was continued for 3 days.
With cooling a saturated solution of NaHCO3 was added until
the vigorous reaction ceased. After addition of 150 mL of
EtOAc the mixture was stirred vigorously for 30 min. The
layers were separated, and the aqueous layer was extracted
three times with EtOAc. After washing with water and drying
(Na2SO4), the solvent was removed in vacuo and the residue
purified by chromatography (SiO2; EtOAc/CH2Cl2, 3:1) to give
yield 48%; light-yellow crystals; mp 245-249 °C (MeOH); H
NMR 3.79 (s, 3H, -OCH3), 3.84 (s, 3H, -OCH3), 6.60-8.00
4
3
(m, 1H, Ar-H), 6.64 (d, J ) 2 Hz, 1H, H-7), 6.97 (dd, J ) 9
Hz, 4J ) 2 Hz, 1H, H-5), 7.02/7.58 (AA′BB′, 3J ) 9 Hz, 4H,
Ar-H), 7.95 (d, 3J ) 8 Hz, 1H, H-4), 9.96 (s, 1H, formyl-H).
Anal. (C17H15NO3) C, H, N.
3-For m yl-4,7-dim eth oxy-2-(4-m eth oxyph en yl)in dole (3f):
1
yield 33%; light-yellow crystals; mp 79-81 °C; H NMR 3.84
(s, 3H, -OCH3), 3.91 (s, 6H, -OCH3), 6.60-7.80 (m, 1H, Ar-
3
H), 6.63 (s, 2H, H-5, H-6), 6.97/7.75 (AA′BB′, J ) 9 Hz, 4H,
Ar-H), 10.62 (s, 1H, formyl-H). Anal. (C18H17NO4) C, H, N.
3-F or m yl-5,6-d im et h oxy-2-(4-m et h oxyp h en yl)in d ole
(3g): yield 12%; light-yellow crystals; mp 268-272 °C; 1H NMR
3.89 (s, 3H, -OCH3), 3.92 (s, 6H, -OCH3), 6.90-7.80 (m, 1H,
Ar-H), 6.98 (s, 1H, H-7), 7.71 (s, 1H, H-4), 7.17/7.69 (AA′BB′,
3J ) 9 Hz, 4H, Ar-H), 9.94 (s, 1H, formyl-H). Anal.
(C18H17NO4) C, H, N.
1
a gray amorphous solid: mp 252-256 °C; H NMR (CD3OD)
3,4-Difor m yl-5,6-d im et h oxy-2-(4-m et h oxyp h en yl)in -
d ole was formed as a byproduct in 8% yield and separated
from the main product by chromatography (SiO2; CH2Cl2/
EtOH, 1:1); white crystals; mp 131-134 °C (MeOH); 1H NMR
3.75 (s, 3H, -OCH3), 3.82 (s, 6H, -OCH3), 7.00-7.35 (m, 1H,
6.64-7.74 (m, 4H, H-7, N-H, O-H), 6.78 (dd, 3J ) 9 Hz, 4J )
2 Hz, 1H, H-6), 6.94/7.52 (AA′BB′, 3J ) 9 Hz, 4H, Ar-H), 7.66
(d, 4J ) 2 Hz, 1H, H-4), 9.82 (s, 1H, formyl-H). Anal.
(C16H11NO3) C; H: calcd, 4.38; found, 5.12. N, calcd, 5.53;
found, 4.78.
3
Ar-H), 7.03/7.43 (AA′BB′, J ) 9 Hz, 4H, Ar-H), 7.25 (s, 1H,
H-7), 7.43 (s, 1H, formyl-H), 8.77 (s, 1H, formyl-H). Anal.
(C19H17NO5) C, H, N.
3-F or m yl-6-h yd r oxy-2-p h en ylin d ole (4b) was prepared
from 3b in 95% yield by a method similar to that described
for 4a : off-white powder; mp 235-237 °C; 1H NMR (acetone-
3-F or m yl-5,7-d im et h oxy-2-(4-m et h oxyp h en yl)in d ole
1
3
3
(3h ): yield 10%; white crystals; mp 170-172 °C (EtOH); H
d6) 6.83 (d, J ) 9 Hz, 1H, H-5), 8.21 (d, J ) 9 Hz, 1H, H-4),
7.32-8.11 (m, 7H, Ar-H), 9.95 (s, 1H, formyl-H). Anal.
(C15H11NO2) H, N; C: calcd, 75.94; found, 75.51.
NMR 3.75 (s, 3H, -OCH3), 3.79 (s, 3H, -OCH3), 3.89 (s, 3H,
-OCH3), 6.40-7.65 (m, 1H, Ar-H), 6.42 (d, 4J ) 2 Hz, 1H,
4
3
H-6), 7.23 (d, J ) 2 Hz, 1H, H-4), 7.04/7.56 (AA′BB′, J ) 9
Hz, 4H, Ar-H), 9.81 (s, 1H, formyl-H). Anal. (C18H17NO4) H,
N; C: calcd, 69.44; found, 68.89.
3-For m yl-6-h yd r oxy-2-(4-h yd r oxyp h en yl)ben zo[b]th io-
p h en e (4c) was prepared from 3n in 89% yield by a method
similar to that described for 4a : white powder; mp 242-246
1
3
3,4-Difor m yl-5,7-d im et h oxy-2-(4-m et h oxyp h en yl)in -
d ole was formed as a byproduct in 7% yield and separated
from the main product by chromatography (SiO2; CH2Cl2/
EtOH, 5:1): yellow crystals; mp 223-225 °C (EtOH); 1H NMR
3.42 (s, 3H, -OCH3), 3.56 (s, 3H, -OCH3), 3.68 (s, 3H, -OCH3),
6.25-7.30 (m, 1H, Ar-H), 6.30 (s, 1H, H-6), 6.61/7.21 (AA′BB′,
3J ) 9 Hz, 4H, Ar-H), 9.80 (s, 1H, formyl-H), 10.27 (s, 1H,
formyl-H). Anal. (C19H17NO5) C, H, N.
6-F lu or o-3-for m yl-2-(4-m eth oxyp h en yl)in d ole (3i): 55%
yield; light-brown crystals; mp 238-240 °C (MeOH); 1H NMR
4.06 (s, 3H, -OCH3), 7.08-7.32 (m, 3H, Ar-H), 7.32/7.90
(AA′BB′, 3J ) 9 Hz, 4H, Ar-H), 7.96-8.25 (m, 1H, H-4), 10.11
(s, 1H, formyl-H). Anal. (C16H12NO2F) C, H, N.
°C; H NMR 7.05/7.65 (AA′BB′, J ) 9 Hz, 4H, Ar-H), 7.10-
7.75 (m, 3H, Ar-H), 8.41 (s, 1H, -OH), 9.82 (s, 1H, -OH),
9.75 (s, 1H, formyl-H). Anal. (C15H10SO3) H; C: calcd, 66.64;
found, 66.02.
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). The monoclonal antibody anti-
R-tubulin (clone DM1A, IgG1 isotype) was obtained from
Sigma and the Cy3-linked anti-mouse-Ig goat antibody from
J ackson Immuno Research Laboratories Inc. Buffer solu-
tions: PEM, 0.1 M PIPES-NaOH, 1 mM EGTA, 1 mM MgSO4,
pH 6.6-6.7; PEMG, 0.1 M PIPES-NaOH, 0.8 M monosodium
L-glutamate, 1 mM EGTA, 1 mM MgSO4, pH 6.6-6.7; PBS,
8.0 g/L NaCl, 0.2 g/L KCl, 1.0 g/L Na2HPO4‚H2O, 0.2 g/L
KH2PO4. Scintillation liquid: Rotiszint Eco Plus (Roth, Karls-
ruhe, 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,
approximately 10% of the transferred volume). After centrifu-
gation at 37 °C for 45 min (20000g) the pellets were weighed
3-F or m yl-5-m eth oxy-2-(4-m eth oxyp h en yl)-1-m eth ylin -
d ole (3j): 45% yield; white crystals; mp 158-160 °C (MeOH);
1H NMR 3.60 (s, 3H, N-CH3), 3.78 (s, 3H, -OCH3), 3.83 (s,
3H, -OCH3), 6.56-7.68 (m, 7H, Ar-H), 9.33 (s, 1H, formyl-
H). Anal. (C18H17NO3) C, H, N.
3-F or m yl-6-m eth oxy-2-(4-m eth oxyp h en yl)-1-m eth ylin -
d ole (3k ): 25% yield; white crystals; mp 143-145 °C (MeOH);
IR (KBr) 1665; 1H NMR 3.70 (s, 3H, N-CH3), 3.91 (s, 6H,
4
3
4
-OCH3), 6.85 (d, J ) 2 Hz, 1H, H-7), 7.10 (dd, J ) 9 Hz, J
) 2 Hz, 1H, H-5), 8.04 (d, 3J ) 9 Hz, 1H, H-4), 7.17/7.54
(AA′BB′, 3J ) 9 Hz, 4H, Ar-H), 9.50 (s, 1H, formyl-H). Anal.
(C18H17NO3) C, H, N.
3-F or m yl-6-m eth oxy-2-(4-m eth oxyp h en yl)-1-p en tylin -
d ole (3l): 44% yield; red crystals; mp 92-94 °C; 1H NMR
0.53-1.79 (m, 9H, CH3-CH2-CH2-CH2-), 3.83 (s, 6H, -OCH3),
4.12 (t, 3J ) 8 Hz, 2H, N-CH2-), 6.67-8.18 (m, 2H, Ar-H),

Methoxy-Substituted 3-Formyl-2-phenylindoles
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 4971
and suspended in ice-cold PEM buffer solution (3 mL/g) and
homogenized in a Teflon-in-glass potter. After standing in ice
for 30 min, the suspension was centrifuged at 2 °C for 30 min
(40000g). The supernatant was separated 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 microtubules were obtained as shiny gellike 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.
Tu bu lin P olym er iza tion Assa y. The pellet of frozen
microtubules 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 M) and
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.
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.
the mixture was kept on ice. Each reaction mixture (0.1 mL)
was filtered under reduced vacuum through a stack of three
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 quantified in
a liquid scintillation counter. Reaction mixtures without
tubulin gave the background values.
F lu or escen ce Micr oscop y. MCF-7 breast cancer cells in
high dilution were plated on sterile glass slides. After incuba-
tion at 37 °C overnight the medium (EMEM with 10% FCS)
was replaced by one containing the drug in appropriate
concentrations. Incubation was continued for another 60 min.
Cells were fixed by consecutive treatments with MeOH and
acetone for 5 min at -20 °C. The fixed cells were washed
thoroughly with PBS. Nonspecific binding sites were blocked
by addition of 90 µL of goat serum onto each slide which was
covered with a coverslip and incubated for 30 min at 37 °C.
After addition of 100 µL of anti-R-tubulin solution (1:500 in
PBS containing 1% BSA) and removal of the coverslips,
incubation was continued for another 60 min. After several
washings with PBS cells were stained with anti-mouse IgG-
Cy3 (1:150 in PBS/1% BSA) for 1 h at 37 °C and treated with
5% propyl gallate in glycerol to reduce fading. Finally, the
coverslips were mounted on the slides and sealed with nail
varnish. The slides were examined with
a fluorescence
microscope (Axioskop with Plan Neofluar 100x/1.3 oil; Zeiss)
equipped with a fluorescence filter BP546/12LP590. Photo-
graphs were taken with a MC100 camera (Zeiss) using Agfa
100 ASA film.
Ack n ow led gm en t . The authors wish to thank
Renate Liebl for excellent technical assistance. This
study was performed in conjunction with the Arbeits-
gemeinschaft Wirkstoffentwicklung in der Onkologie
(AWO) of the German Cancer Society.
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