Antimitotic activities of 2-phenylindole-3-carbaldehydes in human breast cancer cells

Article abstract of DOI:10.1016/j.bmc.2007.05.030

Small molecules such as indoles are attractive as inhibitors of tubulin polymerization. Thus a number of 2-phenylindole-3-carbaldehydes with lipophilic substituents in both aromatic rings was synthesized and evaluated for antitumor activity in MDA-MB 231

Full text of DOI:10.1016/j.bmc.2007.05.030

Bioorganic & Medicinal Chemistry 15 (2007) 5122–5136
Antimitotic activities of 2-phenylindole-3-carbaldehydes
in human breast cancer cells
´
Doris Kaufmann, Michaela Pojarova, Susanne Vogel, Renate Liebl, Robert Gastpar,
Dietmar Gross, Tsuyuki Nishino, Tobias Pfaller and Erwin von Angerer*
Institut fu¨r Pharmazie, Universita¨t Regensburg, D-93040 Regensburg, Germany
Received 23 January 2007; revised 7 May 2007; accepted 11 May 2007
Available online 17 May 2007
Abstract—Small molecules such as indoles are attractive as inhibitors of tubulin polymerization. Thus a number of 2-phenylindole-
3-carbaldehydes with lipophilic substituents in both aromatic rings was synthesized and evaluated for antitumor activity in MDA-
MB 231 and MCF-7 breast cancer cells. Some 5-alkylindole derivatives with a 4-methoxy group in the 2-phenyl ring strongly inhibit
the growth of breast cancer cells with IC50 values of 5–20 nM. Their action can be rationalized by the cell cycle arrest in G2/M phase
due to the inhibition of tubulin polymerization.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
The biological importance of microtubules makes them
an interesting target for the development of anticancer
drugs. For systematic studies on the use of antimitotic
agents in cancer therapy molecules with simple struc-
tures are very attractive because they allow extensive
chemical modifications. Examples are stilbenes,2 aryl-
substituted heterocycles,3 anthracenones,4 benzophe-
nones,5, and analogues6 to name only a few. A variety
of synthetic antimitotic compounds are based on the
indole structure.7–10 In Figure 1, some aryl-substituted
indoles such as 1,11–13 2,14 3,15 and 416 are presented.
Three of these examples carry a trimethoxyphenyl ring
which is considered as important for binding to the
colchicine site on tubulin.
The microtubule system is essential for a number of cel-
lular functions including mitosis and cell replication,
maintenance of cell shape, cellular transport, and motil-
ity. Microtubules are hollow fibers formed by the poly-
merization of a- and b-tubulin heterodimers. The
formation of microtubules and their depolymerization
is a dynamic process which can be interrupted by both
stabilization of microtubules and inhibition of polymer-
ization. A large number of natural products are known
to shift the dynamic equilibrium of the microtubule sys-
tem to one or the other side and abrogate the biological
functions of microtubules thereby. The taxanes and
some other natural products such as epothilones stabi-
lize the microtubule structures, whereas other agents
such as colchicine, combretastatin A-4, and the vinca
alkaloids inhibit the polymerization of a-/b-tubulin di-
mers.1 Some of the natural products are characterized
by complex chemical structures which makes synthesis
and chemical modifications difficult. Others such as
combretastatin A-4 are based on rather simple scaffolds
which can easily be modified.2
An interesting aspect in the application of combretasta-
tin A-4 and related agents in cancer chemotherapy is
their antivascular effect.17 Since microtubules of the
cytoskeleton play a major role in maintaining cell shape,
the elongated endothelial cells of the tumor neovascula-
ture are particularly sensitive to drugs that depolymerize
microtubules and degrade the cytoskeleton. In contrast
to drugs that target tumor angiogenesis compounds
such as combretastatin A-4 phosphate disrupt the
already formed tumor vasculature.18
In a previous study we discovered that some methoxy-
substituted 2-phenylindole-3-carbaldehydes strongly
inhibit the growth of human breast cancer cells.19 Inves-
tigations on the mode of action revealed that the micro-
tubules are the primary target of these agents. In an
Keywords: Phenylindoles; Breast cancer; Tubulin polymerization; Cell
cycle arrest.
*
Corresponding author. Tel.: +49 941 9434821; fax: +49 941
9434820; e-mail: erwin.von-angerer@chemie.uni-regensburg.de
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.030

D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
5123
derivatives were studied for their inhibitory effects on
tubulin polymerization to confirm previous findings. A
representative example was used to investigate morpho-
logical changes of the cells by confocal laser microscopy.
MeO
MeO
NHCOCH3
OH
OMe
MeO
MeO
Combretastatin A-4
MeO
OMe
O
OMe
Colchicine
2. Results and discussion
2.1. Chemistry
OMe
OMe
O
R
For the systematic variation of the substitution pattern
in both aromatic rings a versatile route of synthesis
was required. An appropriate procedure for the syn-
thesis of the substituted 2-phenylindoles 11 and 12
was the Bischler method which involved the condensa-
tion of a substituted aniline 9 with an a-bromophena-
cyl derivative 10 (Fig. 2). Subsequently, the formyl
group was introduced in position 3 of the indole by
a Vilsmeier reaction with DMF and POCl3 to give
the 2-phenylindole-3-carbaldehydes 5 and 6. The reac-
tion of the aldehydes with methylamine and hydroxyl-
amine, respectively, afforded the corresponding imines
7 and 8.
OMe
N
N
H
O
MeO
MeO
H
1 BPR0L075
2
OMe
OMe
OMe
MeO
MeO
OMe
N
H
N
O
Me
N
N
MeO
H
H
2.2. Anti-proliferative activity
4
3
All of the compounds synthesized were first evaluated for
cytostatic activity using hormone-independent human
MDA-MB 231 breast cancer cells in a microplate assay.
Based on previous investigations with methoxy-substi-
tuted 2-phenylindole-3-carbaldehydes, first, a series of
compounds with a methoxy group in the para-position
of the phenyl ring and a variety of lipophilic substituents
in positions 5 and/or 6 of the indole including halogens
and alkyl groups of variable length were tested. Vincris-
tine was used as reference drug in most of the assays.
All 2-(4-methoxyphenyl)indole derivatives strongly
inhibited the growth of MDA-MB 231 cells with IC50 val-
ues below 1 lM (Table 1). The presence of fluorine in the
indole moiety gave only rise to a positive effect when
located in 6-position (5c). The cytostatic potency is
strongly influenced by the length and the structure of
the alkyl substituent in 5-position. It increases with the
length up to 5 carbon atoms and decreases with ramifica-
tion as demonstrated for the butyl derivatives 5j–l. The
lowest IC50 values (5.5–7.4 nM) were found for the
n-butyl (5j), n-pentyl (5m), and n-hexyl (5n) derivatives.
These values are close to the one of vincristine (4.5 nM).
H
O
H
R4
O
R1
R2
R1
R2
R3
OMe
N
H
N
H
R1, R2 = H, alkyl, Cl, OMe
R3 = H, alkyl, F, OMe, CF3
R4 = H, OH, OMe
5
R1, R2 = H, alkyl, F, Cl,
6
Figure 1. Chemical structures of combretastatin A-4, colchicine, and
of some indole-based synthetic inhibitors of tubulin polymerization.
assay with bovine tubulin a marked inhibition on tubu-
lin polymerization was observed. These indole deriva-
tives were also shown to inhibit the binding of
colchicine to tubulin. These findings prompted us to
study this class of compounds in more detail. Since these
derivatives of 2-phenylindole lack structural features
considered typical for inhibitors of tubulin polymeriza-
tion, for example, the 3,4,5-trimethoxyphenyl group
which is characteristic for colchicine, combretastatin
A-4, and podophyllotoxin, the structural requirements
for activity had to be elaborated by a systematic varia-
tion of the substituents in both aromatic rings. Since
the first investigations showed that these substituents
should be lipophilic in nature, alkyl groups of variable
length were introduced. Halogen atoms and the trifluo-
romethyl group completed the spectrum of substituents.
The 1-position of the five-membered ring was kept
unsubstituted because the N–H element of the indole
is essential for the anti-proliferative activity.19 All deriv-
atives were tested for cytostatic activity. Since the inter-
action with the tubulin system usually results in an
arrest of the cell cycle in the G2/M-phase, the most
potent compounds were submitted to a FACS analysis
to record the cell cycle distribution. A small number of
Subsequently the substituent in the phenyl ring was
modified (Table 2). Shifting of the methoxy group to
the meta-position (6a) or introduction of an additional
methoxy (6b) or hydroxyl (6c) group into this position
strongly reduced the activity. A similar decrease in activ-
ity was observed when methoxy was replaced by a
n-butyl (6i) or a fluoro (6j) substituent. Replacement
of the 4-methoxy group by smaller groups such as
methyl, ethyl or trifluoromethyl had only a minor effect
on the potency. The lowest IC50 value (7.8 nM) was
recorded for the 4-methyl derivative 6e. The comparison
of derivatives with n-butyl and ethyl as substituents (6h
and 6i) revealed that the indole moiety tolerates larger
lipophilic substituents than the phenyl ring.

5124
D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
R4
CH2Br
R1
R1
R2
R4
R3
a
R3
O
+
R2
N
NH2
H
11, 12
9
10
H
H
R4
O
NCH3
R1
R2
R1
R2
b
c (5b,j,m, 6k)
d (5j, 6k)
R3
R3
N
N
H
H
7a-d
5a-n, 6a-m
H
NOH
H9C4
R1, R2 = H, alkyl, F, Cl, OCH3
R3 = H, alkyl, F, OCH3, CF3
R4 = H, OH, OCH3
R3
N
H
8a,b
Figure 2. Syntheses of 2-phenylindole-3-carbaldehydes 5 and 6, and of derivatives 7 and 8. Reagents and conditions: (a) xylene, N,N-dimethylaniline,
170 ꢁC; (b) POCl3, DMF, 15 ! 70 ꢁC, 3 h; (c) MeNH2, EtOH, 40 ꢁC, 16 h; (d) H3NOH+Clꢀ, H2O/EtOH, NaOAc, reflux, 3 h.
whereas the formation of oximes (8a and 8b) reduced
Table 1. Anti-proliferative activities of 2-(4-methoxyphenyl)indole-3-
carbaldehydes 5a–n on human MDA-MB 231 and MCF-7 breast
cancer cells
the anti-proliferative activity by one order of magnitude
(Table 3). This difference in bioactivity can be rational-
ized by the higher rate of hydrolysis of the methyl imines
in comparison to the oximes. Hydrolysis experiments
with 7b and 8a followed by HPLC analysis revealed that
7b undergoes significant hydrolysis to the aldehyde 5j at
37 ꢁC, whereas 8a remains stable under these conditions.
O
H
R1
OMe
R2
N
H
All 2-phenylindole derivatives were also tested for anti-
proliferative activity in MCF-7 breast cancer cells
(Tables 1–3). Though similar activities were observed
in both cell lines a tendency to higher IC50 values in
MCF-7 cells was noticed especially for the two oximes
8a and 8b (Table 3).
Compound
R1
R2
MDA-MB 231a
IC50 (nM)
MCF-7b
IC50 (nM)
5a
5b
OMe
H
H
260
35
180
160
43
OMe
F
5c
5d
H
59
F
H
540
27
240
65
5e
H
Me
Cl
Cl
H
2.3. Cell cycle arrest in G2/M-phase
5f
5g
26
86
62
Me
Pr
140
54
Previous studies with 2-phenylindole-3-carbaldehydes
suggested that these aldehydes exert their cytotoxic
effects via tubulin as the intracellular target. Both the
inhibition of tubulin polymerization and stabilization
of microtubules can lead to an arrest of the cell cycle
in the G2/M-phase. Thus, a selection of representative
derivatives including aldehydes, methyl imines, and
oximes was examined for their effects on the cell cycle
progression with MDA-MB 231 cells. The cell cycle-
dependent DNA content was determined by flow cytom-
etry using propidium iodide in permeabilized cells. All
compounds were tested in different concentrations and
compared with vincristine as reference drug. Figure 3
shows the typical decrease of the peak for cells in the
G1/G0 phase and the parallel increase of the number
of cells in G2/M phase for compound 5n. This derivative
and vincristine are rather similar in potency. Only at the
lowest concentration (10 nM) the vinca alkaloid proved
to be somewhat more active (Fig. 4a).
5h
5i
H
20
29
i-Pr
n-Bu
sec-Bu
t-Bu
n-Pent
n-Hex
H
97
22
5j
5k
H
6.7
72
H
180
580
20
5l
5m
H
280
5.5
7.4
4.5
H
5n
Vincristine
H
6.0
n.d.c
a Inhibition of cell growth determined after incubation for 4 days and
subsequent crystal violet staining of viable cells. Mean values of two-
independent experiments with 16–24 replicates, SD are less than 20%.
b Analogous experiment as described for MDA-MB 231 cells with one
exception: the incubation period was 5 days.
c Not determined.
Additional modifications concerned the aldehyde func-
tion. Conversion to the methylamines (7a–d) did not af-
fect the activity in comparison to the free aldehyde,

D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
5125
Table 2. Anti-proliferative activities of 2-phenylindole-3-carbaldehydes 6a–m on human MDA-MB 231 and MCF-7 breast cancer cells
O
R4
H
R1
R2
R3
N
H
Compound
R1
R2
R3
R4
MDA-MB 231a IC50 (nM)
MCF-7b IC50 (nM)
5b
6a
H
OMe
OMe
OMe
OMe
OMe
Cl
OMe
H
H
35
1030
270
800
31
160
390
20
H
OMe
OMe
OH
H
6b
6c
H
OMe
OMe
Me
H
1650
100
37
6d
6e
H
H
Me
Me
H
7.8
48
6f
6g
Me
n-Bu
n-Bu
Et
H
H
165
54
H
Me
Et
H
34
27
6h
6i
H
H
58
H
n-Bu
F
H
300
350
33
200
200
66
6j
6k
n-Bu
n-Bu
n-Pent
n-Hex
H
H
H
CF3
CF3
CF3
H
6l
6m
H
H
42
43
67
22
H
H
Vincristine
4.5
n.d.c
a Inhibition of cell growth determined after incubation for 4 days and subsequent crystal violet staining of viable cells. Mean values of two-
independent experiments with 16–24 replicates, SD are less than 20%.
b Analogous experiment as described for MDA-MB 231 cells with one exception: the incubation period was 5 days.
c Not determined.
Table 3. Anti-proliferative activities of 3-iminomethyl-2-phenylindoles 7 and 8 on human MDA-MB 231 and MCF-7 breast cancer cells
CH=NR5
1
R
R3
R2
N
H
Compound
R1
R2
R3
R5
MDA-MB 231a IC50 (nM)
MCF-7b IC50 (nM)
7a
7b
7c
7d
8a
8b
H
OMe
H
OMe
OMe
OMe
CF3
Me
Me
Me
Me
OH
OH
34
6
220
27
n-Bu
n-Pent
n-Bu
n-Bu
n-Bu
H
6
21
H
32
40
497
140
212
1660
H
OMe
CF3
H
a Inhibition of cell growth determined after incubation for 4 days and subsequent crystal violet staining of viable cells. Mean values of two-
independent experiments with 16–24 replicates, SD are less than 20%.
b Analogous experiment as described for MDA-MB 231 cells with one exception: the incubation period was 5 days.
All indole derivatives tested were able to block the cell
cycle in G2/M phase (Figs. 4 and 5). Generally, this
blockade was accompanied by the appearance of a sig-
nificant sub-G1 peak (‘debris’) probably due to apopto-
tic processes. The cell cycle arrest occurs at
concentrations which were similar to those observed
for the inhibition of cell growth except for the oxime
8a which required a 10-fold higher concentration
(Fig. 5b).
other antimitotic agents. In order to prove this assump-
tion two aldehydes 5j and 6k, the imine 7b, and the
oxime 8a were tested in a microplate assay to obtain pre-
liminary data on their interaction with tubulin. The pro-
gression of tubulin polymerization was measured
turbidimetrically at 350 nm over 28 min after the tem-
perature had been raised from 2 to 37 ꢁC. At a concen-
tration of 5 lM, the aldehyde 6k and the oxime were
inactive, whereas 5j exerted a minor inhibition (23%)
of tubulin polymerization. However, the imine 7b,
derived from the aldehyde 5j, strongly inhibited tubulin
polymerization. From the curves recorded for various
concentrations (Fig. 6) an IC50 value of 1.2 lM was cal-
culated which was lower than that of colchicine (5 lM)
in this assay.
2.4. Inhibition of tubulin polymerization
A rational explanation for the cell cycle arrest is the
inhibition of tubulin polymerization as it is also
observed for colchicine, combretastatin A-4, and many

5126
D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
cuvettes instead of microplates. This assay is more suit-
able for the determination of IC50 values than the micro-
plate assay because it works with larger volumes and
with a better temperature control. However, it requires
the multi-step isolation of tubulin from fresh calf brains
prior to the experiment. In this assay, the aldehydes 5a–c
and the imine 7a were tested for inhibitory activity on
tubulin polymerization in various concentrations. All
four compounds showed a dose-dependent inhibition
of polymerization with IC50 values of 4.0 (5a), 1.5
(5b), and 1.8 lM (5c and 7a) (Fig. 7). The figures for
the latter three compounds are similar to that of colchi-
cine (1.9 lM). These concentrations are nearly two or-
ders of magnitude higher than those observed for the
anti-proliferative effect and the blockade of cell cycle
progression. However, this discrepancy in the effective
concentrations is generally noticed for antimitotic
agents such as combretastatin A-4 and related
structures.12,13,16,20
2.5. Change of cell morphology
Another method to identify the intracellular target of
the 2-phenylindole-3-carbaldehydes was the histological
examination of cells by fluorescence microscopy. Confo-
cal laser microscopy allows the simultaneous visualiza-
tion of chromatin and microtubules and the analysis
of their spatial arrangement. Untreated U-87 MG hu-
man glioma cells display the normal distribution of
microtubules (Fig. 8, panel A). In dividing cells their
functional role can be demonstrated (Fig. 8A, panels
a–c). Treatment of cells with 10 nM vincristine gave rise
to a disruption of the microtubule network and a loss of
Figure 3. Flow cytometry analysis of cell cycle. MDA-MB 231 breast
cancer cells were exposed to compound 5n and vincristine in various
concentrations for 24 h. The DNA content was quantified by the
standard propidium iodide procedure, as described in Section 4. Cells
are assigned to those in G0/G1- (label b), S- (label c), and G2/M-phase
(label d), and to sub-G1 cells (label a), respectively, according to their
DNA content.
A similar assay was performed with a tubulin prepara-
tion from calf brains using temperature-controlled
b
a
60
sub-G1
G1/G0
S
40
20
G2/M
0
ctrl Vin
10
Vin
5g
5g
5n
10
5n
5n
ctrl
Vin
100
5j
10
5j
50
5j
100 nM
100 100 500
50 100 nM
80
60
c
d
40
20
0
ctrl Vin
100
6k
10
6k
50
6h
10
6h
50 nM
ctrl
Vin
100
6g
10
6g
50
6g
100 nM
Figure 4. Cell cycle distribution of MDA-MB 231 cells treated for 24 h with 2-phenylindole-3-carbaldehydes 5 g, 5n (a), 5j (b), 6k, 6h (c), and 6g (d)
in various concentrations. Vincristine (Vin) was used as reference drug. Percentages of sub-G1 cells and cells in G1/G0-phase, S-phase, and G2/M-
phase are shown. Data refer to a representative experiment out of two.

D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
5127
a
b
60
sub-G1
G1/G0
S
40
20
G2/M
0
ctrl
Vin
7b
7b
7b
7b
ctrl
Vin
8a
8a
8a
8a
100
5
10
50
100 nM
100
50
100
500 1000 nM
Figure 5. Cell cycle distribution of MDA-MB 231 cells treated for 24 h with the methylimine 7b (a) and the oxime 8a (b) in various concentrations.
Vincristine (Vin) was used as reference drug. Percentages of sub-G1 cells and cells in G1/G0-phase, S-phase, and G2/M-phase are shown. Data refer to
a representative experiment out of two.
120
control
10
colchicine
100
-7
7b, 5 x 10
7b, 1 x 10
7b, 2 x 10
7b, 5 x 10
M
M
M
M
-6
-6
-6
80
60
40
20
0
8
6
4
2
0
colchicine
5a
5b
5c
7a
-6
-5
10
10
Concentration [mol/l]
Figure 7. Inhibitory effects of various 2-phenylindole derivatives 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.
0
5
10
15
20
25
30
Time [min]
Figure 6. Effect of methylimine 7b on tubulin assembly. Optical
density at 355 nm was measured every minute over a period of 28 min
simultaneously for all compounds and concentrations after tempera-
ture had been switched from 2 to 37 ꢁC. Control wells contained
tubulin and the solvent (0.1% DMSO). The concentration of colchicine
was 5 lM.
shown that substituents in positions 1, 4, and 7 of the in-
dole and 3 in the phenyl ring had a detrimental effect on
the anti-proliferative activity. Thus, only substituents in
5- and 6-position of the indole and in the para-position
of the phenyl ring were modified. The most favorable
substitution pattern comprises an alkyl chain of 4 to 6
carbon atoms and a methoxy or a methyl group in the
phenyl ring. Though a number of synthetic indole-based
inhibitors of tubulin polymerization are known (Fig. 1)7
the structure of the 2-phenylindole-3-carbaldehydes is
unique because it possesses an aldehyde function and
lacks the 3,4,5-trimethoxyphenyl ring which is typical
for the majority of indole-based inhibitors of tubulin
its function in the mitotic process (Fig. 8, panel B).
When the cells were treated with the aldehyde 5j
(50 nM) the microtubules condensed around the nucleus
and were no longer capable of separating the chromo-
somes for mitosis (Fig. 8, panel C). The nucleus showed
chromatin condensation and disintegration of the nucle-
ar matrix probably due to an apoptotic cell death
(Fig. 8C, panel b).21
polymerization.11–13,15,16,22,23 Other indoles with
carbonyl function are the 2-aroylindoles.14
a
2.6. Discussion
The aim of this study was to identify the most favorable
substitution pattern of the 2-phenylindole-3-carbaldehy-
des for antimitotic activity. Since hydrophilic substitu-
ents such as hydroxy functions have been shown to
reduce the antitumor effect dramatically19 only lipophilic
groups were considered. Preliminary investigations have
Though the 2-phenylindole-3-carbaldehydes can be con-
sidered as stilbene analogues they differ from combre-
tastatin A-4 by the trans arrangement of the phenyl
rings. This may explain the lack of cytotoxicity of the
4,5,6-trimethoxy-2-(4-methoxyphenyl)indole-3-carbal-

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D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
Figure 8. Effects of vincristine and 2-phenylindole-3-carbaldehyde 5j on the organization of cellular microtubule network and nuclear structure of
U-87 MG human glioblastoma cells, visualized by confocal laser fluorescence microscopy. (Panel A) Untreated control cells which undergo normal
mitoses as shown in panels a, b, and c (a, stained tubulin; b, stained nucleus; c, merged images). (Panel B) Vincristine (10 nM) leads to a disturbed and
partially degraded tubulin network that has lost its function (a, b, c). (Panel C) Indole 5j (50 nM) causes a condensation of microtubules around the
nucleus and a loss of function (a, b, c). Some of the nuclei desintegrate upon treatment (b).
dehyde which is furnished with the 3,4,5-trimethoxyphe-
nyl group (data not shown). Also the low activity found
with 6c which possesses the same substitution pattern as
that of the second phenyl ring of combretastatin A-4 is
in agreement with these considerations. The results of
this and other studies suggest that the colchicine binding
site tolerates a large variety of different chemical struc-
tures as long as they contain two aromatic moieties with
appropriate substituents.2,3
From the data presented the primary site of action
appears to be the a/b-tubulin heterodimer whose poly-
merization is strongly inhibited by the aldehydes tested.
This action results in the destruction of the microtubule
network and abrogates its function. This action can be
visualized by fluorescence microscopy as demonstrated
for the aldehyde 5j. Already at a concentration of
50 nM the microtubules condense around the nucleus
and the filament structure is lost. At higher concentra-

D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
5129
tions most of the microtubules have disappeared.19 This
change is accompanied by a structural alteration of the
nucleus which desintegrates probably due to apoptotic
processes.
Varian MAT-311A spectrometer at 70 eV. Purity of all
compounds was checked by TLC. Elemental analyses of
crystalline compounds were performed by the Mikroanal-
ytisches Laboratorium, University of Regensburg. The
syntheses of 5-methoxy-2-(4-methoxyphenyl)indole-3-
carbaldehyde (5a), 6-methoxy-2-(4-methoxyphenyl)indole-
3-carbaldehyde (5b), and 6-fluoro-2-(4-methoxyphenyl)-
indole-3-carbaldehyde (5c) have been described
previously.19
Interesting results were obtained from the comparison of
the aldehydes with the derivatives that possess an imine
structure. In the cytotoxicity assays all of the methyl
imines showed activities identical to those of the corre-
sponding aldehydes which can be rationalized by the
hydrolysis of the imines. The hydrolytic conversion of
the imine to the parent aldehyde was confirmed by HPLC
analysis. A significant difference was observed when
experiments such as the inhibition of tubulin polymeriza-
tion last only for a short period of time. The imine 7b
proved to be much more potent as inhibitor of tubulin
polymerization than the corresponding aldehyde 5j. This
difference may be due to a higher binding affinity of the
imine for tubulin compared to the aldehyde, but this
assumption has not yet been proved. The oximes which
are shown to be resistant to hydrolysis at 37 ꢁC are less
active than the corresponding aldehydes by one order of
magnitude. This difference is reflected by their inactivity
in the tubulin polymerization assay (data not shown).
4.2. Preparation of 2-phenylindoles 11 and 12
A solution of 40(30)-substituted 2-bromoacetophenone
(26 mmol) in xylene (150 mL) was added dropwise to a
solution of the substituted aniline (66 mmol) in N,N-
dimethylaniline (15 mL) at 170 ꢁC over a period of 1 h.
The reaction mixture was stirred at this temperature
for 3–24 h. After cooling, the dark brown solution was
poured into 400 mL of a 2 N HCl/EtOAc mixture
(1:1). The aqueous phase was extracted twice with
EtOAc (200 mL). The organic phase was washed with
sat. NaCl solution and dried over Na2SO4. The solvent
was removed under vacuum and the product was puri-
fied by column chromatography (SiO2) with different
mobile phases on the basis of dichloromethane and sub-
sequent crystallization.
3. Conclusion
4.2.1. 5-Fluoro-2-(4-methoxyphenyl)indole (11d). Color-
1
This study revealed that the 2-phenylindole-3-carbalde-
hydes are an interesting class of compounds with high
anti-proliferative activity in two breast cancer cell lines.
We were able to show that tubulin is the primary target
of these agents which inhibit the polymerization of tubu-
lin to functional microtubules by binding to the colchi-
cine binding site. This interaction with tubulin leads to
cell cycle arrest in the G2/M phase and probably leads
to an apoptotic cell death. The in vitro potencies of some
of the aldehydes are in same range as those of vincristine
and combretastatin A-4. Preliminary investigations on
the in vivo activity, however, showed that these alde-
hydes do not inhibit the growth of transplanted murine
tumors. One of the possible reasons might be the instabil-
ity of the aldehyde function toward metabolic reactions.
Insufficient bioavailability could be another reason. In
order to overcome this problem we are going to modify
the carbonyl function to improve the metabolic stability
of this essential structural element. Two of these modifi-
cations, conversion of the aldehydes to methyl imines
and oximes, respectively, are included in this study.
Though both types of imines are active, their mode of
action seems to be different. Results from other modifica-
tions will be reported in due time.
less crystals (21% yield), mp 215 ꢁC. H NMR (DMSO-
d6) d 3.83 (s, 3H, –OCH3); 6.81 (d, 4J = 2.0 Hz, 1H,
indole-H3); 6.88 (dd, 4J = 2.2 Hz, 4J = 2.0 Hz, 1H,
indole-H4); 7.03, 7.77 (AA0BB0, J = 8.8 Hz, 4H, phe-
3
nyl-H); 7.23 (d, J = 8.0 Hz, 1H, indole-H7); 7.33 (dd,
3
3J = 8.0 Hz, J = 2.2 Hz, 1H, indole-H6); 11.50 (s, 1H,
4
N–H). Anal. for C15H12FNO; calcd C, 74.68; H, 5.01;
N, 5.81; found C, 74.39; H, 5.08; N, 5.77.
4.2.2. 6-Chloro-2-(4-methoxyphenyl)indole (11e). Green-
ish solid (71% yield), mp 198 ꢁC (EtOH). 1H NMR
(CDCl3) d 3.86 (s, 3H, –OCH3); 6.64 (s, 1H, indole-
H3); 7.07, 7.49 (AA0BB0, 3J = 9 Hz, 4H, phenyl-H);
7.25 (dd, J = 8 Hz, J = 2 Hz, 1H, indole-H5); 7.49 (d,
3
4
3
4J = 2 Hz, 1H, indole-H7); 7.59 (d, J = 8 Hz, 1H, in-
dole-H4); 8.32 (s, br, 1H, N–H). Anal. for C15H12ClNO;
calcd C, 69.91; H, 4.69; N, 5.46; found C, 69.12; H, 4.48;
N, 5.54.
4.2.3. 6-Chloro-2-(4-methoxyphenyl)-5-methylindole (11f).
The preparation afforded two products, 11f (22% yield)
and isomeric 4-chloro-2-(4-methoxyphenyl)-5-methylin-
dole (14% yield), which were separated by column chro-
matography. The main product (11f) was obtained as
1
colorless solid, mp 198 ꢁC (EtOH). H NMR (DMSO-
d6) d 2.37 (s, 3H, –CH3); 3.81 (s, 3H, –OCH3); 6.70 (s,
3
4. Experimental
1H, indole-H3); 7.03, 7.76 (AA0BB0, J = 9 Hz, 4H, phe-
nyl-H); 7.36 (s, 1H, indole-H4); 7.43 (s, 1H, indole-H7);
11.43 (s, 1H, N–H). Anal. for C16H14ClNO; calcd C,
70.72; H, 5.19; N, 5.15; found C, 70.49; H, 5.21; N, 5.10.
4.1. General methods
Melting points were determined on a Buchi 510 appara-
¨
tus and are uncorrected. 1H NMR spectra were recorded
on a Bruker AC-250 spectrometer with TMS as internal
standard and were in accord with the assigned struc-
tures. Mass spectra (PI-EI MS) were measured on a
4.2.4. 2-(4-Methoxyphenyl)-5-methylindole (11g). Color-
1
less crystals (30% yield), mp 237 ꢁC (EtOH). H NMR
(CDCl3) d 2.46 (s, 3H, –CH3); 3.88 (s, 3H, –OCH3);
6.66 (s, 1H, indole-H3); 7.00–7.61 (m, 3H, indole-

5130
D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
3
H4,6,7); 7.00, 7.61 (AA0BB0, J = 9 Hz, 4H, phenyl-H);
8.19 (s, br, 1H, N–H). Anal. for C16H15NO; calcd C,
80.98; H, 6.37; N, 5.90; found C, 80.46; H, 6.11; N, 5.80.
NMR (DMSO-d6) d 0.89 (t, 3J = 7 Hz, 3H, –CH2–
CH3); 1.34 (m, 2H, –CH2–CH2–CH3); 1.66 (m, 2H,
–CH2–CH2–CH2–); 2.69 (t, 3J = 7 Hz, 2H, –CH2–
4
CH2–); 3.85 (s, 3H, –OCH3); 6.64 (d, J = 1 Hz, 1H, in-
4.2.5. 2-(4-Methoxyphenyl)-5-n-propylindole (11h). Color-
less crystals (20% yield), mp 195–196 ꢁC (EtOH). 1H
NMR (CDCl3) d 0.96 (t, 3J = 7 Hz, 3H, –CH2–CH3);
1.68 (sext, 3J = 7 Hz, 3H, –CH2–CH2–CH3); 2.67 (t,
3J = 7 Hz, 3H, –CH2–CH2–); 3.84 (s, 3H, –OCH3); 6.64
dole-H3); 6.94–7.01 (m, 1H, indole-H6); 6.96, 7.57
3
3
(AA0BB0, J = 9 Hz, 4H, Phenyl-H); 7.26 (d, J = 8 Hz,
1H, indole-H7); 7.39 (s, 1H, indole-H4); 8.13 (s, br,
1H, –NH). Anal. for C20H23NO; calcd C, 81.87; H,
7.90; N, 4.77; found C, 82.05; H, 7.79, N, 5.33.
(d, J = 1 Hz, 1H, indole-H3); 6.94–7.01 (m, 1H, indole-
4
H6); 6.96, 7.26 (AA0BB0, J = 9 Hz, 4H, phenyl-H); 7.26
4.2.11. 5-n-Hexyl-2-(4-methoxyphenyl)indole (11n). White
solid (14% yield), mp 197–198 ꢁC (EtOH). 1H NMR
3
(d, J = 7 Hz, 1H, indole-H7); 7.39 (s, 1H, indole-H4);
3
3
8.13 (s, br, 1H, N–H). Anal. for C18H19NO; calcd C,
81.48; H, 7.22, N, 5.28; found C, 81.23; H, 7.32; N, 5.18.
(CDCl3) d 0.88 (t, J = 7 Hz, 3H, –CH2–CH3); 1.32 (m,
2H, –CH2–CH2–CH3); 1.65 (m, 2H, –CH2–CH2–CH2–);
2.17 (m, 4H, –CH2–(CH2)2–CH2–); 2.71 (t, 3J = 7 Hz,
2H, –CH2–CH2–); 3.85 (s, 3H, –OCH3), 6.64 (d,
5J = 1 Hz, 1H, indole-H3); 6.94–7.01 (m, 1H, indole-H6);
4.2.6. 5-Isopropyl-2-(4-methoxyphenyl)indole (11i). Col-
orless crystals (14% yield), mp 203 ꢁC (EtOH). 1H
NMR (DMSO-d6) d 1.24 (d, 3J = 7 Hz, 6H, –CH–
3
6.97, 7.57 (AA0BB0, J = 9 Hz, 4H, phenyl-H); 7.27 (d,
3
(CH3)2); 2.93 (sept, J = 7 Hz, 1H, –CH–(CH3)2); 3.80
3J = 8 Hz, 1H, indole-H7); 7.39 (s, 1H, indole-H4); 8.20
(s, br, 1H, –NH). Anal. for C21H25NO; calcd C, 82.05; H,
8.20; N, 4.56; found C, 82.04; H, 7.85; N, 4.42.
(s, 3H, –OCH3); 6.68 (d, 1H, 4J = 2 Hz, indole-H3);
6.96 (dd, 4J = 2 Hz, 3J = 8 Hz, 1H, indole-H6); 7.01,
7.76 (AA0BB0, 3J = 9 Hz, 4H, phenyl-H); 7.26 (d,
4J = 2 Hz, 1H, indole-H4); 7.30 (d, J = 8 Hz, 1H, in-
4.2.12. 2-(3,4-Dimethoxyphenyl)-6-methoxyindole (12b).
3
dole-H7); 11.26 (s, br, 1H, N–H). Anal. for
C19H21NO; calcd C, 81.48; H, 7.22; N, 5.28; found C,
80.34; H, 7.07; N, 5.18.
Colorless crystals (15% yield), mp 161 ꢁC (EtOH). H
1
NMR (CDCl3) d 3.85 (s, 3H, –OCH3); 3.92 (s, 3H,
–OCH3); 3.96 (s, 3H, –OCH3); 6.65 (s, 1H, indole-H3);
6.79 (dd, J = 7 Hz, J = 2 Hz, 1H, ArH); 6.88–7.15 (m,
3
5
3
4.2.7. 5-n-Butyl-2-(4-methoxyphenyl)indole (11j). Color-
less crystals (76% yield), mp 228 ꢁC (EtOH). 1H
NMR (DMSO-d6) d 0.94 (t, J = 7 Hz, 3H, –CH2–CH3);
4H, indole-H4,5,7, ArH); 7.47 (d, J = 7 Hz, 1H, ArH);
8.20 (s, br, 1H, N–H). Anal. for C17H17NO3; calcd C,
72.07; H, 6.05; N, 4.94; found C, 71.84; H, 5.99; N, 4.90.
3
3
1.38 (sext, J = 7 Hz, 2H, –CH2–CH2–CH3); 1.65 (quin,
3J = 7 Hz, 2H, –CH2–CH2–CH2–); 2.70 (t, 3J = 7 Hz,
2H, –CH2–CH2–); 3.85 (s, 3H, –OCH3); 6.63 (d, 1H,
4J = 2 Hz, indole-H3), 6.94–7.01 (m, 1H, indole-H6);
4.2.13. 2-(3-Hydroxy-4-methoxyphenyl)-6-methoxyindole
(12c). The title compound was not isolated, but used
immediately for the next step of synthesis.
3
6.96, 7.57 (AA0BB0, J = 9 Hz, 4H, phenyl-H); 7.26 (d,
3J = 8 Hz, 1H, indole-H7); 7.38 (s, 1H, indole-H4); 8.14
(s, br, 1H, –NH). Anal. for C19H21NO; calcd C, 81.68;
H, 7.58; N, 5.01; found C, 81.51; H, 7.61; N, 4.97.
4.2.14. 6-Methoxy-2-(4-methylphenyl)indole (12d). White
solid (42% yield), mp 162 ꢁC (EtOH). 1H NMR (CDCl3)
d 2.40 (s, 3H, –CH3); 3.88 (s, 3H, –OCH3); 6.72 (s, br,
1H, indole-H3); 6.81 (dd, 3J = 8 Hz, 4J = 2 Hz, 1H,
indole-H5); 6.92 (s, 1H, indole-H7); 7.26, 7.50 (AA0BB0,
3J = 9 Hz, 4H, phenyl-H); 7.24–7.32 (m, 1H, indol-H4),
8.24 (s, br, 1H, N–H); MS m/z (%) 238 (16, [MH]Å+),
237 (90, MÅ+), 222 (100, [MꢀÅCH3]+), 194 (12,
[MꢀÅ+CO–CH3]+).
4.2.8. 5-sec-Butyl-2-(4-methoxyphenyl)indole (11k). Col-
orless crystals (38% yield), mp 191 ꢁC (EtOH). 1H
NMR (DMSO-d6) d 0.85 (t, 3J = 7 Hz, 3H, –CH2–
3
CH3); 1.29 (d, J = 7 Hz, 3H, –CH–CH3); 1.65 (quin,
3J = 7 Hz, 2H, –CH2–CH3); 2.67 (t, 3J = 7 Hz, 1H,
–CH–CH3); 3.85 (s, 3H, –OCH3); 6.65 (s, 1H, indole-
H3); 6.95-7.02 (m, 1H, indole-H6); 6.97, 7.57 (AA0BB0,
3J = 9 Hz, 4H, phenyl-H); 7.30 (d, 3J = 8 Hz, 1H, in-
dole-H7); 7.39 (s, 1H, indole-H4); 8.15 (s, br, 1H,
–NH). Anal. for C19H21NO; calcd C, 81.68; H, 7.58;
N, 5.01; found C, 81.51; H, 7.60; N, 4.94.
4.2.15. 6-Chloro-2-(4-methylphenyl)indole (12e). Orange
solid (40% yield), mp 145 ꢁC (EtOH). 1H NMR
4
(DMSO-d6) d 2.34 (s, 3H, –CH3); 6.87 (d, J = 1 Hz,
1H, indole-H3); 7.00 (dd, 3J = 8 Hz, 4J = 2 Hz, 1H,
3
indole-H5); 7.28, 7.52 (AA0BB0, J = 9 Hz, 4H, phenyl-
H); 7.38 (d, 4J = 2 Hz, 1H, indole-H7); 7.51 (d,
3J = 8 Hz, 1H, indole-H4); 11.64 (s, 1H, N–H). Anal.
for C15H12ClN; calcd C, 74.53; H, 5.00; N, 7.79; found
C, 74.46; H, 5.02; N, 5.61.
4.2.9. 5-tert-Butyl-2-(4-methoxyphenyl)indole (11l). White
crystals (36% yield), mp 224 ꢁC (EtOH). 1H NMR
(DMSO-d6) d 1.24 (s, 9H, ꢀ(CH3)3); 3.80 (s, 3H,
–OCH3); 6.70 (d, J = 2 Hz, 1H, indole-H3); 7.02, 7.76
4
3
5
(AA0BB0, J = 9 Hz, 4H, phenyl-H); 7.14 (dd, J = 2 Hz,
3J = 8 Hz, 1H, indole-H6); 7.29 (d, 3J = 8 Hz, 1H, indole-
H7); 7.45 (d, 4J = 2 Hz, 1H, indole-H4); 11.25 (s, 1H,
–NH). Anal. for C19H21NO; calcd C, 81.68; H, 7.58; N,
5.01; found C, 81.31; H, 7.57; N, 4.91.
4.2.16. 5-Methyl-2-(4-methylphenyl)indole (12f). White
solid (36% yield), mp 227 ꢁC (EtOH). 1H NMR (CDCl3)
d 2.38 (s, 3H, –CH3); 2.44 (s, 3H, –CH3); 6.69 (s, 1H, in-
dole-H3); 6.99 (dd, 3J = 8 Hz, 4J = 1 Hz, 1H, indole-H6);
7.24, 7.53 (AA0BB0, 3J = 9 Hz, 4H, phenyl-H); 7.20–7.27
(m, 2H, indole-H4,7); 8.19 (s, br, 1H, N–H). Anal. for
C16H15N; calcd C, 86.82; H, 6.83; N, 6.35; C, 85.94;
H, 6.68; N, 6.21.
4.2.10. 2-(4-Methoxyphenyl)-5-n-pentylindole (11m).
White solid (21% yield), mp 197–198 ꢁC (EtOH). 1H

D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
5131
4.2.17. 5-Butyl-2-(4-methylphenyl)indole (12g). Orange
(CH2)2–CH3); 1.66 (m, 2H, –CH2–CH2–CH2–); 2.70
(t, 2H, J = 7 Hz, –CH2–CH2–); 6.68 (d, 1H, J = 2 Hz,
1
3
4
powder (15% yield), mp 181–182 ꢁC. H NMR (CDCl3)
d 0.94 (t, 3H, 3J = 7 Hz, –CH2–CH3); 1.39 (m, 2H,
–CH2–CH2–CH3); 1.65 (m, 2H, –CH2–CH2–CH2–);
2.37 (s, 3H, –CH3); 2.70 (t, 2H, 3J = 7 Hz, –CH2–
indole-H4); 7.07 (dd, 1H, J = 8 Hz, J = 2 Hz, indole-
3
4
H6); 7.32 (d, 1H, 3J = 8 Hz, indole-H7); 7.43 (s, 1H,
3
indole-H3); 7.74, 7.68 (AA0BB0, 4H, J = 8 Hz, phenyl-
H); 8.29 (s, br, 1H, N–H).
4
5
CH2–); 6.70 (dd, 1H, J = 2 Hz, J = 1 Hz, indole-H4);
7.01 (dd, 1H, 3J = 8 Hz, 4J = 2 Hz, indole-H6); 7.22
(m, 3H, indole-H7 and phenyl-H); 7.40 (s, 1H, indole-
4.2.23. 5-Hexyl-2-[4-(trifluoromethyl)phenyl]indole (12m).
Orange powder (31% yield). The compound was directly
3
H3); 7.51 (d, 2H, J = 8 Hz, phenyl-H); 8.16 (s, br, 1H,
1
N–H). Anal. for C19H21N; calcd C, 86.64; H, 8.04; N,
5.32; found C, 86.31; H, 7.65; N, 5.34.
used in the following reaction step. H NMR (CDCl3) d
0.88 (t, 3H, J = 7 Hz, –CH2–CH3); 1.36 (m, 4H, –CH2–
3
(CH2)2–CH3); 1.65 (m, 4H, –CH2–(CH2)2–CH2–); 2.70
(t, 2H, J = 7 Hz, –CH2–CH2–); 6.87 (d, 1H, J = 2 Hz,
3
4
4.2.18. 5-Butyl-2-(4-ethylphenyl)indole (12h). Yellow
1
3
4
powder (18% yield), mp 167–168 ꢁC. H NMR (CDCl3)
indole-H4); 7.07 (dd, 1H, J = 8 Hz, J = 2 Hz, indole-
3
d 0.93 (t, 3H, 3J = 7 Hz, –CH2–CH3); 1.26 (t, 3H,
3J = 7 Hz, –CH2–CH3); 1.38 (m, 2H, –CH2–CH2–
CH3); 1.65 (m, 2H, –CH2–CH2–CH2–); 2.68 (m, 4H,
H6); 7.33 (d, 1H, J = 8 Hz, indole-H7); 7.43 (s, 1H, in-
3
dole-H3); 7.74, 7.67 (AA0BB0, 4H, J = 8 Hz, phenyl-H);
8.30 (s, br, 1H, N–H).
4
–CH2–CH3 and –CH2–CH2–); 6.71 (d, 1H, J = 2 Hz,
3
4
indole-H4); 7.01 (dd, 1H, J = 8 Hz, J = 2 Hz, indole-
4.3. Preparation of 2-phenylindole-3-carbaldehydes 5 and 6
H6); 7.27 (m, 3H, phenyl-H and indole-H7); 7.40 (s,
1H, indole-H3); 7.57 (d, 2H, J = 8 Hz, phenyl-H); 8.21
Under nitrogen, POCl3 (10 mmol) was added dropwise
to DMF (10 mmol) at 15 ꢁC. The mixture was stirred
for 15 min, then the solution of the respective 2-pheny-
lindole (1 mmol) in DMF (50 mL) was added dropwise
to the mixture, which was then heated to 70 ꢁC. After
3 h of heating, the reaction mixture was diluted with
ice water (200 mL), neutralized with 40% NaOH, and
extracted with chloroform. The chloroform extract was
washed with water and dried over NaSO4. The solvent
was removed under vacuum. Crystallization of the resi-
due from methanol gave the pure product.
3
(s, br, 1H, N–H). Anal. for C20H23N; calcd C, 86.59;
H, 8.36; N, 5.05; found C, 86.55; H, 8.07; N, 5.03.
4.2.19. 2-(4-Butylphenyl)-5-ethylindole (12i). Orange
1
powder (20% yield), mp 183–184 ꢁC. H NMR (CDCl3)
d 0.90 (t, 3H, 3J = 7 Hz, –CH2–CH3); 1.21 (t, 3H,
3J = 7 Hz, –CH2–CH3); 1.32 (m, 2H, –CH2–CH2–
CH3); 1.58 (m, 2H, –CH2–CH2–CH2); 2.63 (m, 4H,
–CH2–CH3 and –CH2–CH2–); 6.74 (d, 1H, 4J = 2 Hz, in-
3
4
dole-H4); 7.04 (dd, 1H, J = 8 Hz, J = 2 Hz, indole-H6);
7.31 (d, 1H, J = 8 Hz, indole-H7); 7.43 (s, 1H, indole-
3
H3); 7.56, 7.24 (AA0BB0, 4H, 3J = 8 Hz, phenyl-H);
8.22 (s, br, 1H, N–H). Anal. For C20H23N; calcd C,
86.59; H, 8.36; N, 5.05; found C, 86.10; H, 8.33; N, 4.93.
4.3.1. 5-Fluoro-2-(4-methoxyphenyl)indole-3-carbaldehyde
1
(5d). Beige crystals (85% yield), mp 250 ꢁC. H NMR
(DMSO-d6) d 3.86 (s, 3H, –OCH3); 7.05–7.13 (m, 1H, in-
dole-H); 7.15, 7.72 (AA0BB0, 3J = 8.7 Hz, 4H, phenyl-H);
7.44–7.89 (m, 1H, indole-H); 7.81–7.89 (m, 1H, indole-H);
9.92 (s, 1H, –CHO); 12.39 (s, 1H, N–H). Anal. for
C16H12FNO; calcd C, 71.37; H, 4.49; N, 5.20; found C,
71.05; H, 4.39; N, 5.08.
4.2.20. 5-Butyl-2-(4-fluorophenyl)indole (12j). Orange
1
powder (48% yield), mp 140–142 ꢁC. H NMR (CDCl3)
d 0.94 (t, 3H, 3J = 7 Hz, –CH2–CH3); 1.45 (m, 2H,
–CH2–CH2–CH3); 1.65 (m, 2H, –CH2–CH2–CH2); 2.70
(t, 2H, 3J = 7 Hz, –CH2–CH2–); 6.69 (1, 1H, indole-
H4); 7.03 (dd, 1H, 3J = 8 Hz, 4J = 2 Hz, indole-H6);
4.3.2. 6-Chloro-2-(4-methoxyphenyl)indole-3-carbaldehyde
(5e). Gray solid (28% yield), mp 260 ꢁC. 1H NMR
(DMSO-d6) d 3.86 (s, 3H, –OCH3); 7.16, 7.73 (AA0
BB0, 3J = 9 Hz, 4H, phenyl-H); 7.25 (dd, 3J = 7 Hz,
3
7.29 (d, 1H, J = 8 Hz, indole-H7); 7.41 (s, 1H, indole-
H3); 7.65, 7.13 (m, 4H, phenyl-H); 8.16 (s, br, 1H,
–NH–). Anal. for C18H18FN; calcd C, 80.96; H, 6.78;
N, 5.24; found C, 80.12; H, 6.65; N, 5.11.
4
4J = 2 Hz, 1H, indole-H5); 7.49 (d, J = 2 Hz, 1H, in-
dole-H7); 8.16 (d, 3J = 7 Hz, 1H, indole-H4); 9.95 (s, 1H,
–CHO); 12.40 (s, 1H, N–H). Anal. for C16H12ClNO2:
calcd C, 67.26; H, 4.23; N, 4.90; found C, 67.48; H, 4.44;
N, 4.76.
4.2.21. 5-Butyl-2-[4-(trifluoromethyl)phenyl]indole (12k).
1
Yellow powder (31% yield), mp 120–121 ꢁC. H NMR
(CDCl3) d 0.94 (t, 3H, 3J = 7 Hz, –CH2–CH3); 1.38
(m, 2H, –CH2–CH2–CH3); 1.70 (m, 2H, –CH2–CH2–
CH2–); 2.71 (t, 2H, 3J = 7 Hz, –CH2–CH2–); 6.85 (d,
1H, 4J = 2 Hz, indole-H4); 7.07 (dd, 1H, 3J = 8 Hz,
4J = 2 Hz, indole-H6); 7.33 (d, 1H, 3J = 8 Hz, indole-
H7); 7.43 (s, 1H, indole-H3); 7.74, 7.67 (AA0BB0, 4H,
3J = 8 Hz, phenyl-H); 8.29 (s, br, 1H, N–H). Anal. for
C19H18F3N; calcd C, 71.91; H, 5.72; N, 4.41; found C,
71.56; H, 5.85; N, 4.15.
4.3.3. 6-Chloro-2-(4-methoxyphenyl)-5-methylindole-3-carb-
aldehyde (5f). Gray solid (28% yield), mp 287 ꢁC. 1H NMR
(DMSO-d6) d 2.43 (s, 3H, –CH3); 3.33 (s, 3H, –OCH3);
7.16, 7.73 (AA0BB0, 3J = 9 Hz, 4H, phenyl-H); 7.49 (s,
1H, indole-H4); 8.14 (s, 1H, indole-H7); 9.92 (s, 1H,
–CHO); 12.31 (s, br, 1H, N–H). Anal. for C17H14ClNO2;
calcd C, 68.12; H, 4.71; N, 4.67; found C, 67.82; H, 4.85;
N, 4.68.
4.2.22. 5-Pentyl-2-[4-(trifluoromethyl)phenyl]indole (12l).
Orange powder (32% yield). The compound was directly
used in the following reaction step. 1H NMR (CDCl3) d
0.90 (t, 3H, 3J = 7 Hz, –CH2–CH3); 1.34 (m, 4H, –CH2–
4.3.4. 2-(4-Methoxyphenyl)-5-methylindole-3-carbaldehyde
(5g). Gray solid (79% yield), mp 242 ꢁC. 1H NMR
(DMSO-d6) d 2.42 (s, 3H, –CH3); 3.86 (s, 3H, –OCH3);

5132
D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
3
7.09 (dd, J = 9 Hz, 4J = 1 Hz, 1H, indole-H6); 7.15, 7.71
NMR (DMSO-d6) d 0.87 (t, 3J = 7 Hz, 3H, –CH2–
CH3); 1.30 (m, 4H, –CH2–(CH2)2–CH3); 1.62 (m, 2H, –
CH2–CH2–CH2); 2.68 (t, J = 7 Hz, 2H, –CH2–CH2–);
3
3
(AA0BB0, J = 9 Hz, 4H, phenyl-H); 7.36 (d, J = 8 Hz,
1H, indole-H7); 8.01 (s, 1H, indole-H4); 9.92 (s, 1H,
–CHO); 12.17 (s, br, 1H, N–H). Anal. for C17H15NO2;
calcd C, 76.96; H, 5.70; N, 5.28; found C, 76.57; H,
5.65; N, 5.17.
3
3.86 (s, 3H, –OCH3); 7.10 (dd, 3J = 8 Hz, 4J = 2 Hz,
1H, indole-H6); 7.16, 7.70 (AA0BB0, 3J = 9 Hz, 4H, phe-
3
nyl-H); 7.38 (d, J = 8 Hz, 1H, indole-H7); 8.00 (s, 1H,
indole-H4); 9.92 (s, 1H, –CHO); 12.18 (s, 1H, N–H);
Anal. for C21H23NO2; calcd C, 78.47; H, 7.21; N, 4.36;
found C, 77.97; H, 6.70; N, 4.25.
4.3.5. 2-(4-Methoxyphenyl)-5-n-propylindole-3-carbalde-
1
hyde (5h). Colorless solid (35% yield), mp 209 ꢁC. H
3
NMR (DMSO-d6) d 0.91 (t, J = 7 Hz, 3H, –CH2–CH3);
1.63 (sext, 3J = 7 Hz, 2 H, –CH2–CH2–CH3); 2.67 (t,
3J = 7 Hz, 3H, –CH2–CH2–); 3.86 (s, 3H, –OCH3), 7.10
4.3.11. 5-n-Hexyl-2-(4-methoxyphenyl)indole-3-carbalde-
1
hyde (5n). Orange crystals (60% yield), mp 176 ꢁC. H
(dd, J = 2 Hz, J = 8 Hz, 1H, indole-H6), 7.15 und 7.70
NMR (DMSO-d6) d 0.88 (t, 3J = 7 Hz, 3H, –CH2–
CH3); 1.29 (m, 6H, –CH2–(CH2)3–CH3); 1.61 (m, 2H,
–CH2–CH2–CH2–); 2.68 (t, 3J = 7 Hz, 2H, –CH2–
4
3
3
3
(AA0BB0, J = 9 Hz, 4H, phenyl-H), 7.38 (d, J = 8 Hz,
1H, indole-H7), 8.01 (d, J = 2 Hz, 1H, indole-H4), 9.93
4
3
(s, 1H, –CHO), 12.17 (s, 1H, N–H). Anal. for
C19H19NO2; calcd C, 77.96; H, 6.53; N, 4.77; found C,
77.55; H, 6.47; N, 4.80.
CH2–); 3.86 (s, 3H, –OCH3); 7.10 (d, J = 8 Hz, 1H, in-
dole-H6); 7.15, 7.70 (AA0BB0, 3J = 9 Hz, 4H, phenyl-H);
3
7.37 (d, J = 8 Hz, 1H, indole-H7); 8.00 (s, 1H, indole-
H4); 9.92 (s, 1H, –CHO); 12.17 (s, 1H, N–H). Anal.
for C22H25NO2; calcd C, 78.77; H, 7.51; N, 4.18; found
C, 77.95; H, 7.57; N, 4.25.
4.3.6. 5-Isopropyl-2-(4-methoxyphenyl)indole-3-carbaldehyde
1
(5i). Colorless solid (54% yield), mp 217 ꢁC. H NMR
3
(DMSO-d6) d 1.25 (d, J = 7 Hz, 6H, –CH–(CH3)2); 2.99
(sept, J = 7 Hz, 1H, –CH–(CH3)2); 3.85 (s, 3H, –OCH3);
3
4.3.12. 6-Methoxy-2-(3-methoxyphenyl)indole-3-carbalde-
hyde (6a). White crystals (80% yield), mp 190 ꢁC. 1H
NMR (DMSO-d6) d 3.80 (s, 3H, –OCH3); 3.85 (s, 3H,
–OCH3); 6.80-8.10 (m, 7H, ArH); 9.94 (s, 1H, –CHO);
12.19 (s, 1H, N–H). Anal. for C17H15NO3; calcd C,
72.58; H, 5.37; N, 4.98; found C, 71.97; H, 5.60; N, 4.61.
3
7.15, 7.69 (AA0BB0, J = 9 Hz, 4H, phenyl-H); 7.16 (d,
3J = 8 Hz, 1H, indole-H6); 7.38 (d, J = 8 Hz, 1H, indole-
3
H7); 8.05 (s, 1H, indole-H4); 9.92 (s, 1H, –CHO); 12.16
(s, 1H, N–H). Anal. for C19H19NO2; calcd C, 77.96; H,
6.53; N, 4.77; found C, 77.57; H, 6.45; N, 4.81.
4.3.7. 5-n-Butyl-2-(4-methoxyphenyl)indole-3-carbalde-
4.3.13. 2-(3,4-Dimethoxyphenyl)-6-methoxyindole-3-carbal-
dehyde (6b). Yellow crystals (55% yield), mp 210 ꢁC. H
1
1
hyde (5j). Orange crystals (44% yield), mp 210 ꢁC. H
NMR (DMSO-d6) d 0.92 (t, 3J = 7 Hz, 3H, –CH2–CH3);
NMR (DMSO-d6) d 3.81 (s, 3H, –OCH3); 3.85 (s, 3H,
–OCH3); 3.87 (s, 3H, –OCH3); 6.87 (dd, 3J = 9 Hz,
4J = 2 Hz, 1H, phenyl-H6); 6.95 (d, 4J = 2 Hz, 1H, in-
dole-H7); 7.15 (d, 3J = 8 Hz, 1H, indole-H5); 7.28 (d,
4J = 2 Hz, 1H, phenyl-H2); 7.27-7.32 (m, 1H, phenyl-H5);
3
1.34 (sext, J = 7 Hz, 2H, –CH2–CH2–CH3); 1.63 (quin,
3J = 7 Hz, 2H, –CH2–CH2–CH2); 2.73 (t, 3J = 7 Hz, 2H,
–CH2–CH2–); 3.89 (s, 3H, –OCH3); 7.22-7.24 (m, 1H, in-
dole-H6); 7.21, 7.67 (AA0BB0, 3J = 9 Hz, 4H, phenyl-H);
7.22–7.24 (m, 1H, indole-H6); 7.52 (d, 3J = 8 Hz, 1H, in-
dole-H7); 7.57 (s, 1H, indole-H4), 8.71 (s, 1H, –CHO);
13.19 (s, 1H, N–H). Anal. for C20H21NO2; calcd C,
78.15; H, 6.89; N, 4.56; found C, 78.08; H, 6.83; N, 4.66.
3
8.05 (d, J = 8 Hz, 1H, indole-H4); 9.95 (s, 1H, –CHO);
12.06 (s, br, 1H, N–H). Anal. for C18H17NO4; calcd C,
69.44; H, 5.50; N, 4.50; found C, 69.23; H, 5.58; N, 4.22.
4.3.14. 2-(3-Hydroxy-4-methoxyphenyl)-6-methoxyindole-
3-carbaldehyde (6c). Colorless crystals (57% yield), mp
4.3.8. 5-sec-Butyl-2-(4-methoxyphenyl)indole-3-carbalde-
hyde (5k). Slightly violet solid (68% yield), mp 179 ꢁC.
1
253 ꢁC. H NMR (DMSO-d6) d 3.81 (s, 3H, –OCH3);
1H NMR (DMSO-d6) d 0.80 (t, J = 7 Hz, 3H, –CH2–
3.88(s, 3H, –OCH3);6.85(dd, 3J = 9 Hz, 4J = 2 Hz, 1H, in-
dole-H5); 6.96–6.97 (m, 2H, phenyl-H2,5); 7.16 (dd,
3J = 8 Hz, 4J = 2 Hz, 1H, phenyl-H6); 7.28 (dd, 4J = 2 Hz,
3
3
CH3); 1.25 (d, J = 7 Hz, 3H, –CH–CH3); 1.61 (quin,
3J = 7 Hz, 2H, –CH2–CH3); 2.69 (t, 3J = 7 Hz, 1H,
–CH–CH3); 3.86 (s, 3H, –OCH3); 7.10–7.17 (m, 1H,
3
1H, indole-H7); 8.03 (d, J = 9 Hz, 1H, indole-H4); 9.54
3
indole-H6), 7.15, 7.70 (AA0BB0, J = 9 Hz, 4H, phenyl-
(s, 1H, –OH); 9.93 (s, 1H, –CHO); 11.99 (s, 1H, N–H).
Anal. for C17H15NO4; calcd C, 68.68; H, 5.08, N, 4.71;
found C, 68.53; H, 5.02; N, 4.65.
H); 7.39 (d, 3J = 8 Hz, 1H, indole-H7); 8.02 (s, 1H,
indole-H4); 9.92 (s, 1H, –CHO); 12.14 (s, 1H, N–H).
Anal. for C20H21NO2; calcd C, 78.15; H, 6.89; N, 4.56;
found C, 77.81; H, 6.86; N, 4.51.
4.3.15. 6-Methoxy-2-(4-methylphenyl)indole-3-carbaldehyde
1
(6d). Colorless crystals (60% yield), mp 250 ꢁC. H NMR
4.3.9. 5-tert-Butyl-2-(4-methoxyphenyl)indole-3-carbalde-
(DMSO-d6) d 2.42 (s, 3H, –CH3); 3.81 (s, 3H, –OCH3);
1
3
4
hyde (5l). Colorless solid (67% yield), mp 223 ꢁC. H
6.87 (dd, J = 8 Hz, J = 2 Hz, 1H, indole-H5); 6.95 (d,
3
NMR (DMSO-d6) d 1.36 (s, 9H, –CH3); 3.86 (s, 3H,
–OCH3); 7.15, 7.69 (AA0BB0, 3J = 9 Hz, 4H, phenyl-
H); 7.36–7.42 (m, 2H, indole-H6,7); 8.23 (s, 1H, indole-
H4); 9.93 (s, 1H, –CHO); 12.13 (s, 1H, N–H); MS: m/z
(%) = 307 (61, M Å+), 292 (100, [MꢀÅCH3]+).
4J = 2 Hz, 1H, indole-H7); 7.40, 7.63 (AA0BB0, J = 9 Hz,
4H, phenyl-H); 8.05 (d, J = 8 Hz, 1H, indole-H4); 9.91
3
(s, 1H, –CHO); 12.17 (s, 1H, N–H). Anal. for
C17H15NO2; calcd C, 76.96; H, 5.70; N, 5.28; found C,
76.24; H, 5.89; N, 4.91.
4.3.10. 2-(4-Methoxyphenyl)-5-n-pentylindole-3-carbalde-
hyde (5m). Orange crystals (60% yield), mp 182 ꢁC. H
4.3.16.
6-Chloro-2-(4-methylphenyl)indole-3-carbalde-
1
hyde (6e). Colorless crystals (57% yield), mp 268 ꢁC.

D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
5133
1H NMR (DMSO-d6) d 2.42 (s, 3H, –CH3); 7.26 (dd,
3J = 8 Hz, 4J = 2 Hz, 1H, indole-H5); 7.42, 7.68
(AA0BB0, J = 9 Hz, 4H, phenyl-H); 7.50 (d, J = 2 Hz,
1H, indole-H7); 8.18 (d, 3J = 9 Hz, 1H, indole-H4);
9.96 (s, 1H, –CHO); 12.46 (s, 1H, N–H). Anal. for
C16H12ClNO; calcd C, 71.25; H, 4.48; N, 5.19; found
C, 71.45; H, 4.63; N, 4.86.
H4); 9.87 (s, 1H, –CHO); 12.30 (s, br, 1H, N–H); MS:
m/z (%) = 295 (64, [M]+O); 252 (100, [M]+O–OC3H7).
Anal. for C19H18FNO; calcd C, 77.27; H, 6.14; N, 4.74;
found C, 76.40; H, 6.10; N, 4.60.
3
4
4.3.22. 5-n-Butyl-2-[4-(trifluoromethyl)phenyl]indole-3-carb-
aldehyde (6k). Orange powder (45% yield), mp 230–231 ꢁC.
3
1H NMR (DMSO-d6) d 0.91 (t, 3H, J = 7 Hz, –CH2–
CH3); 1.33 (m, 2H, –CH2–CH2–CH3); 1.60 (m, 2H,
4.3.17. 5-Methyl-2-(4-methylphenyl)indole-3-carbaldehyde
(6f). Colorless crystals (48% yield), mp 226 ꢁC. 1H
NMR (DMSO-d6) d 2.41 (s, 3H, –CH3); 2.43 (s, 3H,
3
–CH2–CH2–CH2); 2.70 (t, 2H, J = 7 Hz, –CH2–CH2–);
3
4
7.17 (dd, 1H, J = 8 Hz, J = 2 Hz, indole-H6); 7.44 (d,
3
4
3
–CH3); 7.10 (dd, J = 8 Hz, J = 1 Hz, 1H, indole-H6);
1H, J = 8 Hz, indole-H7); 7.95, 8.00 (AA0BB0, 4H,
3
7.40, 7.65 (AA0BB0, J = 9 Hz, 4H, phenyl-H); 7.39 (d,
3J = 8 Hz, phenyl-H); 8.05 (s, 1H, indole-H4); 9.97 (s,
1H, –CHO); 12.48 (s, br, 1H, N–H). Anal. for
C20H18F3NO; calcd C, 69.55; H, 5.25; N, 4.05; found C,
69.14; H, 5.18; N, 4.09.
3J = 8 Hz, 1H, indole-H7); 8.02 (d, 4J = 1 Hz, 1H, in-
dole-H4); 9.94 (s, 1H, –CHO); 12.20 (s, 1H, N–H). Anal.
for C17H15NO; calcd C, 81.90; H, 6.06; N, 5.62; found C,
81.64; H, 5.95; N, 5.38.
4.3.23. 5-Pentyl-2-[4-(trifluoromethyl)phenyl]indole-3-carb-
aldehyde (6l). Yellow powder (32% yield), mp 226–227 ꢁC.
4.3.18. 5-Butyl-2-(4-methylphenyl)indole-3-carbaldehyde (6g).
Orange solid (68% yield), mp 175–178 ꢁC. 1H NMR
3
1H NMR (DMSO-d6) d 0.88 (t, 3H, J = 7 Hz, –CH2–
CH3); 1.31 (m, 4H, –CH2–(CH2)2–CH3); 1.64 (m, 2H,
3
(DMSO-d6) d 0.92 (t, 3H, J = 7 Hz, –CH2–CH3); 1.34
3
–CH2–CH2–CH2–); 2.71 (t, 2H, J = 7 Hz, –CH2–CH2–);
(m, 2H, –CH2–CH2–CH3); 1.61 (m, 2H, –CH2–CH2–
CH2–); 2.41 (s, 3H, –CH3); 2.68 (t, 2H, 3J = 7 Hz,
–CH2–CH2–); 7.13 (dd, 1H, 3J = 8 Hz, 4J = 2 Hz,
indole-H6); 7.41 (m, 3H, indole-H7, phenyl-H); 7.66 (d,
3
4
7.19 (dd, 1H, J = 8 Hz, J = 2 Hz, indole-H6); 7.46 (d,
1H, J = 8 Hz, indole-H7); 7.97, 8.02 (AA0BB0, 4H,
3
3J = 8 Hz, phenyl-H); 8.07 (s, 1H, indole-H4); 9.97 (s, 1H,
–CHO); 12.51 (s, br, 1H, N–H). Anal. for C21H20F3NO;
calcd C, 70.18; H, 5.61; N, 3.89; found C, 70.00; H, 5.49;
N, 3.62.
3
2H, J = 8 Hz, phenyl-H); 8.03 (s, 1H, indole-H4); 9.94
(s, 1H, –CHO); 12.24 (s, br, 1H, N–H); MS: m/z
(%) = 291 (67, [M]+O); 248 (100, [M]+O–OC3H7). Anal.
for C20H21NO; calcd C, 82.44; H, 7.26; N, 4.81; found
C, 81.22; H, 7.07; N, 4.12.
4.3.24. 5-Hexyl-2-[4-(trifluoromethyl)phenyl]indole-3-carb-
aldehyde (6m). Yellow solid (34% yield), mp 224–225 ꢁC.
3
4.3.19. 5-Butyl-2-(4-ethylphenyl)indole-3-carbaldehyde (6h).
1H NMR (DMSO-d6) d 0.85 (t, 3H, J = 7 Hz, –CH2–
CH3); 1.28 (m, 6H, –CH2–(CH2)3–CH3); 1.61 (m, 2H,
Yellow solid (46% yield), mp 178–180 ꢁC. 1H NMR
3
3
–CH2–CH2–CH2–); 2.69 (t, 2H, J = 7 Hz, –CH2–CH2–);
(DMSO-d6) d 0.91 (t, 3H, J = 7 Hz, –CH2–CH3); 1.30
3
4
(m, 5H, –CH2–CH3, –CH2–CH2–CH3); 1.59 (m, 2H,
–CH2–CH2–CH2–); 2.70 (m, 4H, –CH2–CH3, –CH2–
CH2–); 7.11 (dd, 1H, 3J = 8 Hz, 4J = 2 Hz, indole-H6); 7.39
7.16 (dd, 1H, J = 8 Hz, J = 2 Hz, indole-H6); 7.44 (d,
1H, J = 8 Hz, indole-H7); 7.95, 8.00 (AA0BB0, 4H,
3
3J = 8 Hz, phenyl-H); 8.04 (s, 1H, indole-H4); 9.97 (s, 1H,
–CHO); 12.48 (s, br, 1H, N–H). Anal. for C22H22F3NO;
calcd C, 70.76; H, 5.94; N, 3.75; found C, 70.98; H, 6.01;
N, 3.51.
3
(d, 1H, J = 8 Hz, indole-H7); 7.67, 7.43 (AA0BB0, 4H,
3J = 8 Hz, phenyl-H); 8.01 (s, 1H, indole-H4); 9.93 (s,
1H, –CHO); 12.22 (s, br, 1H, N–H); MS: m/z (%) = 305
(90, [M]+O); 262 (100, [M]+O–OC3H7). Anal. for
C21H23NO; calcd C, 82.59; H, 7.59; N, 4.59; found C,
81.38; H, 7.43; N, 4.21.
4.4. Preparation of the carbaldehyde methyl imines 7
To a solution of the 2-phenylindole-3-carbaldehyde 5
or 6 (2 mmol) in CH2Cl2 (100 mL) was added under
nitrogen methylamine (0.01 mol) as 33% solution in
EtOH. The mixture was stirred at 40 ꢁC overnight.
Then, H2O (100 mL) was added and the aqueous
mixture was extracted several times with 50-mL por-
tions of CHCl3. The combined organic layers were
washed with H2O and brine, and dried over
Na2SO4. After evaporation of the solvent in vacuo,
the residue was purified by crystallization from
EtOH.
4.3.20. 2-(4-Butylphenyl)-5-ethylindole-3-carbaldehyde (6i).
Light brown solid (30% yield), mp 175–176 ꢁC. 1H NMR
(DMSO-d6) d 0.91 (t, 3H, 3J = 7 Hz, –CH2–CH3); 1.23 (t,
3
3H, J = 7 Hz, –CH2–CH3); 1.35 (m, 2H, –CH2–CH2–
CH3); 1.61 (m, 2H, –CH2–CH2–CH2–); 2.70 (m, 4H,
–CH2–CH3, –CH2–CH2–); 7.17 (dd, 1H, 3J = 8 Hz,
4J = 2 Hz, indole-H6); 7.40 (m, 3H, indole-H7, phenyl-H);
3
7.66 (d, 2H, J = 8 Hz, phenyl-H); 8.03 (s, 1H, indole-
H4); 9.94 (s, 1H, –CHO); 12.22 (s, br, 1H, N–H). Anal.
for C21H23NO; calcd C, 82.59; H, 7.59; N, 4.59; found C,
81.55; 7.25; N, 4.16.
4.4.1. 6-Methoxy-2-(4-methoxyphenyl)-3-[(methylimino)-
methyl]indole (7a). Yellow resin (85% yield). H NMR
(d6-acetone) d 3.35 (d, J = 1 Hz, 3H, @N–CH3); 3.73
1
4.3.21. 5-Butyl-2-(4-fluorophenyl)indole-3-carbaldehyde (6j).
4
Orange solid (40% yield), mp 192–194 ꢁC. 1H NMR
3
(DMSO-d6) d 0.91 (t, 3H, J = 7 Hz, –CH2–CH3); 1.32
(m, 2H, –CH2–CH2–CH3); 1.60 (m, 2H, –CH2–CH2–
(s, 3H, –OCH3); 3.82 (s, 3H, –OCH3); 6.70 (dd,
3J = 9 Hz, 4J = 2 Hz, 1H, indole-H5); 6.86 (d,
4J = 2 Hz, 1H, indole-H7); 6.99, 7.49 (AA0BB0, 3J =
9 Hz, 4H, phenyl-H); 8.24 (d, 3J = 9 Hz, 1H, indole-
3
CH2); 2.69 (t, 2H, J = 7 Hz, –CH2–CH2–); 7.13 (dd, 1H,
4
3J = 8 Hz, J = 2 Hz, indole-H6); 7.43 (m, 3H, indole-H7,
phenyl-H); 7.82 (d, 2H, phenyl-H); 8.02 (s, 1H, indole-
4
H4); 8.42 (q, J = 1 Hz, 1H, –CH @N–CH3).

5134
D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
4.4.2. 5-n-Butyl-2-(4-methoxyphenyl)-3-[(methylimino)-
methyl]indole (7b). Colorless crystals (67% yield), mp
(s, 1H, –OH), 11.53 (s, 1H, N–H). Anal. for
C20H22N2O2; calcd C, 74.51; H, 6.88; N, 8.69; found
C, 74.67; H, 6.84; N, 8.56.
207–209 ꢁC. 1H NMR (DMSO-d6)
d
0.91 (t,
3
3J = 7.3 Hz, 3H, –CH2 –CH3); 1.33 (sext, J = 7.4 Hz,
2H, –CH2–CH2–CH3); 1.58 (quin, 3J = 7.5 Hz, 2H,
–CH2–CH2–CH2–); 2.65 (t, 3J = 7.6 Hz, 2H, –CH2–
4.5.2. 5-n-Butyl-3-[(hydroxyimino)methyl]-2-[4-(trifluoro-
methyl)phenyl]indole (8b). Colorless crystals (86% yield),
mp 166 ꢁC. 1H NMR (DMSO-d6) d 0.91 (t, 3J = 7.3 Hz,
3H, –CH2–CH3); 1.33 (sext, 3J = 7.4 Hz, 2H, –CH2–
4
CH2–CH2–); 3.40 (d, J = 1.3 Hz, 3H, @N–CH3); 3.84
(s, 3H, –OCH3); 7.01 (dd, J = 8.3Hz, J = 1.6 Hz, 1H,
3
4
3
indole-H6); 7.13, 7.55 (AA0BB0, J = 8.8 Hz, 4H, phe-
CH2–CH3);
1.59
(quin,
3J = 7.5 Hz,
2H,
3
nyl-H); 7.29 (d, J = 8.2 Hz, 1H, indole-H7); 8.09 (d,
–CH2–CH2–CH2–); 2.67 (t, 3J = 7.5 Hz, 2H, –CH2–
4
3
4
4J = 0.9 Hz, 1H, indole-H4); 8.43 (d, J = 1.4 Hz, 1H,
CH2–); 7.10 (dd, J = 8.3 Hz, J = 1.6 Hz, 1H, indole-
H6); 7.37 (d, 3J = 8.3 Hz, 1H, indole-H7); 7.81, 7.92
–CH @NCH3); 11.56 (s, 1H, N–H). Anal. for
C21H24N2O; calcd C, 78.71; H, 7.55; N, 8.74; found C,
78.72; H, 7.55; N, 8.53.
3
(AA0BB0, J = 7.9 Hz, 4H, phenyl-H); 7.94 (s, 1H, in-
dole-H4); 8.29 (s, 1H, –CH@N–); 10.84 (s, 1H, –OH);
11.81 (s, 1H, N–H). Anal. for C20H19F3N2O; calcd C,
66.66; H, 5.31; N, 7.77; found C, 66.71; H, 5.32; N, 7.71.
4.4.3. 5-n-Pentyl-2-(4-methoxyphenyl)-indol-3-[(methylimi-
no)methyl]indole (7c). Colorless crystals (77% yield), mp
1
3
203 ꢁC. H NMR (DMSO-d6) d 0.86 (t, J = 6.8 Hz, 3H,
–CH2–CH3); 1.29 (m, 4H, –CH2–(CH2)2–CH3); 1.59 (m,
2H, –CH2–CH2–CH2); 2.64 (t, J = 7.5 Hz, 2H, –CH2–
4.6. Materials and reagents for bioassays
3
Drugs and biochemicals were obtained from Sigma
(Deisenhofen, Germany). The microplate-based tubulin
polymerization assay (Cytoskeletonꢂ) was purchased
from Tebu-bio (Offenbach, Germany). MDA-MB 231
and MCF-7 breast cancer cells were obtained from the
American Type Culture collection (ATCC, Rockville,
MD, USA). Buffer solutions used: 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-glu-
tamate, 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.
4
CH2–); 3.40 (d, J = 1.1 Hz, 3H, @N–CH3); 3.84 (s, 3H,
3
4
–OMe); 7.01 (dd, J = 8.3 Hz, J = 1.6 Hz, 1H, indole-
H6); 7.13, 7.55 (AA0BB0, 3J = 8.7 Hz, 4H, phenyl-H);
3
7.29 (d, J = 8.2Hz, 1H, indole-H7); 8.09 (s, 1H, indole-
H4); 8.43 (s, 1H, –CH =NCH3); 11.56 (s, 1H, N–H). Anal.
for C22H26N2O; calcd C, 79.01; H, 7.84; N, 8.38; found C,
78.65; H, 7.51; N, 8.27.
4.4.4. 5-n-Butyl-2-[4-(trifluoromethyl)phenyl]-3-[(methyli-
mino)methyl]indole (7d). Colorless solid (63% yield), mp
186 ꢁC. 1H NMR (DMSO-d6) d 0.91 (t, 3J = 7.3 Hz, 3H,
–CH2–CH3); 1.33 (sext, 3J = 7.4 Hz, 2H, –CH2–CH2–
CH3);1.59 (quin, 3J = 7.5 Hz, 2H, –CH2–CH2–CH2–);
2.67 (t, 3J = 7.6 Hz, 2H, –CH2–CH2–), 3.43 (d,
4J = 1.2 Hz, 3H, @N–CH3); 7.08 (dd, 3J = 8.3 Hz,
4J = 1.6 Hz, 1H, indole-H6); 7.36 (d, 3J = 8.3 Hz, 1H, in-
4.7. Determination of anti-proliferative activity
Hormone-independent human MDA-MB 231 breast
cancer cells were grown in McCoy-5a medium, supple-
mented 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 transferred to 96-well microplates
(100 lL/well). After the cells grew for 4 days in a humid-
ified incubator with 5% CO2 at 37 ꢁC, medium was
replaced by one containing the test compounds
(200 lL/well). Control wells (16/plate) contained 0.1%
of DMF that was used for the preparation of stock solu-
tions. Initial cell density was determined by addition of
glutaric dialdehyde (1% in PBS; 100 lL/well) instead of
test compound. After incubation for about 4 days, med-
ium was removed and 100 lL of glutaric dialdehyde in
PBS (1%) was added for fixation. After 15 min, the solu-
tion of aldehyde was decanted. Cells were stained by
treating them for 25 min with 100 lL of an aqueous
solution of crystal violet (0.02%). After decanting, cells
were washed several times with water to remove adher-
ent dye. After addition of 100 lL 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.
3
dole-H7); 7.85, 7.93 (AA0BB0, J = 8.3 Hz, 4H, phenyl-
H); 8.14 (s, 1H, indole-H4); 8.49 (s, 1H, –CH @NCH3);
11.85 (s, 1H, N–H). Anal. for C21H21F3N2; calcd C,
70.37; H, 5.91; N, 7.82; found C, 70.17; H, 5.69; N, 7.04.
4.5. Preparation of the oximes 8
To a solution of hydroxylammonium chloride (1.2 mmol)
in H2O (8 mL), a solution of the 2-phenylindole-3-carb-
aldehyde (1 mmol) in EtOH (8 mL) and NaOAc
(1.6 mmol) were added. The mixture was heated under
reflux for 3 h and stirred at room temperature overnight.
After repeated extraction with CH2Cl2 the combined
organic layers were washed with water and dried
(Na2SO4). The solvent was removed in vacuo and the
residue recrystallized from EtOH.
4.5.1. 5-n-Butyl-3-[(hydroxyimino)methyl]-2-(4-methoxy-
phenyl)indole (8a). Colorless crystals (67% yield), mp
138 ꢁC. 1H NMR (DMSO-d6) d 0.90 (t, 3J = 7.3 Hz,
3H, –CH2–CH3); 1.32 (sext, 3J = 7.3 Hz, 2H, –CH2–
CH2–CH3); 1.58 (quin, 3J = 7.5 Hz, 2H, –CH2–CH2–
3
CH2–); 2.65 (t, J = 7.5 Hz, 2H, –CH2–CH2–); 3.84 (s,
3H, –OCH3); 7.02 (dd, 3J = 8.3 Hz, 4J = 1.5 Hz, 1H,
indole-H6); 7.13, 7.51 (AA0BB0, 3J = 8.7 Hz, 4H,
phenyl-H); 7.30 (d, 3J = 8.2 Hz, 1H, indole-H7); 7.88
(s, 1H, indole-H4); 8.22 (s, 1H, –CH@N–); 10.63
For hormone-sensitive MCF-7 human breast cancer
cells a similar procedure to that described for MDA-
MB 231 cells was applied with modifications: Cells were
grown in EMEM supplemented with sodium pyruvate

D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
5135
(110 mg/L), gentamycin sulfate (50 mg/L), NaHCO3
(2.2 g/L), phenol red, and 10% FCS for 5 days.
tion at 37 ꢁC for 45 min (20,000g) the pellets were
weighed 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 (40,000g). The superna-
tant 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 gel-like pellet.
The yields ranged from 2 to 6 g per brain. Aliquots were
frozen in liquid nitrogen and stored at ꢀ70 ꢁC.
4.8. Flow cytometry
MDA-MB 231 cells were grown to 70–80% confluence
on the bottom of a 75 cm2 culture flask. Test substances
were dissolved in DMF and diluted to the required con-
centrations. The content of DMF in medium and in con-
trols after 1000 times dilution was set to 0.1%. For each
concentration one culture flask was used. Cells were ex-
posed to test substances for 24 h. This incubation time
was necessary to obtain a sufficient number of cells
(5 · 106 cells). After incubation cells were trypsinized,
centrifuged (250g) in an excess of serum containing med-
ium for 10 min, and washed with PBS.
Purity
was
checked
by
polyacrylamide
gel
electrophoresis.
4.10.2. Temperature-induced tubulin polymerization. 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. Depolymeriza-
tion was completed by keeping the mixture at 0 ꢁC for
30 min, followed by centrifugation at 2 ꢁC (30 min;
30,000g) to remove insoluble protein. Each reaction
tube contained 0.46 mL of the supernatant and 20 lL
of the DMSO solution of the test compound in varying
concentrations. Reaction mixtures were preincubated at
37 ꢁC for 15 min and chilled on ice followed by addition
of 20 lL of a 25 mM GTP solution in PEMG buffer to
each tube. Reaction mixtures were transferred to cuv-
ettes of a UV spectrophotometer connected to two dif-
ferent temperature controller. First, the temperature
inside the cuvettes was held at 2 ꢁC. The cuvette holder
was then switched to the second temperature controller
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. Three-independent
experiments were performed for the 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.
The pellets of cells were resuspended in 1 mL of PBS
and 9 mL of 70% EtOH (ice-cold). EtOH and PBS
were removed by centrifugation and the cells were
washed with PBS. After the addition of 0.5 mL of
PBS and 0.5 mL of DNA extraction buffer the cells
were transferred into Eppendorf cups (2 mL), incu-
bated for 5 min at room temperature, and centrifuged
again. The cells were stained with 1 mL of propidium
iodide solution for at least 30 min at room tempera-
ture and then analyzed by flow cytometry using a
FACS-CaliburTM. For analysis, data files for four
parameters were collected for 8000–15,000 events from
each sample. The flow rate was adjusted to ca.
300 cells/s for sufficient accuracy. Data were analyzed
by the WinMDI 2.8 software.
4.9. Tubulin polymerization assay on microplates
This assay was performed according to the vendor’s pro-
tocol. Stock solutions of test compounds in DMSO were
diluted 1:10 with G-PEM buffer. Ten microliters of this
solution was added to the corresponding well of a
microplate which was warmed in a microplate reader
(Tecan) to 37 ꢁC. Ice-cold tubulin was suspended in cold
G-PEM buffer + glycerol (5%) and depolymerized at
0 ꢁC within 1 min. Within 30 s, 100 lL of this solution
was added to the warm solutions of the test compounds
on the microplate and the plate returned to the reader.
Absorbance at 355 nm was recorded at 37 ꢁC for
30 min. Reference drugs were taxoterre and colchicine;
control wells contained only the solvent (1%).
4.11. Confocal laser scanning microscopy
U-87 MG cells were seeded into 8-well Lab-Tek Cham-
ber Slides (Nunc, Wiesbaden, Germany). At 75% conflu-
ence the culture medium was replaced with medium
containing vincristine (10 nM), 2-phenylindole-3-carbal-
dehyde 5j (50 nM), or corresponding vehicle (EtOH).
The cells were incubated at 37 ꢁC for 3 h. After removal
of the culture medium the cells were fixed with 4% para-
formaldehyde solution in PBS for 20 min at room tem-
perature. Thereafter each well was washed three times
with PBS supplemented by 0.5% BSA. For permeabili-
zation cells were incubated with PBS (+0.5% BSA) con-
taining 1% Triton X-100 (Serva, Heidelberg, Germany)
for 10 min at room temperature and washed three times
with PBS (+0.5% BSA).
4.10. Tubulin polymerization assay in cuvettes
4.10.1. Isolation and purification of calf brain tubulin. The
cortex of one or two fresh calf brains in ice-cold PEM
buffer (1 mL/g tissue, +16 mg DTE/100 mL buffer solu-
tion) was homogenized in portions. After centrifugation
(90 min; 20,000g) at 2–4 ꢁC, the supernatant was care-
fully 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 under-
layered with a pre-warmed (37 ꢁC) sucrose solution
(10% in PEM buffer solution containing 1 mM GTP, ap-
prox 10% of the transferred volume). After centrifuga-
Nuclei and chromosomes were stained with SYTOX-
Greenꢂ nucleic acid stain (Molecular Probes, Eugene,
OR, USA). Microtubules were stained using mouse
anti-human a-tubulin primary antibody (Dianova,
Hamburg, Germany) and Cy5-conjugated goat anti-
mouse secondary antibody (Molecular Probes). All

5136
D. Kaufmann et al. / Bioorg. Med. Chem. 15 (2007) 5122–5136
11. Kuo, C. C.; Hsieh, H. P.; Pan, W. Y.; Chen, C. P.; Liou, J.
P.; Lee, S. J.; Chang, Y. L.; Chen, L. T.; Chen, C. T.;
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antibodies were used in a 1:200 dilution in PBS, contain-
ing 0.5% BSA. A Carl Zeiss Axiovert 200M LSM510
confocal laser scanning microscope was employed for
acquisition of fluorescence images.
13. Liou, J. P.; Mahindroo, N.; Chang, C. W.; Guo, F. M.;
Lee, S. W.; Tan, U. K.; Yeh, T. K.; Kuo, C. C.; Chang, Y.
W.; Lu, P. H.; Tung, Y. S.; Lin, K. T.; Chang, J. Y.;
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