New C5-alkylated indolobenzazepinones acting as inhibitors of tubulin polymerization: Cytotoxic and antitumor activities

Article abstract of DOI:10.1021/jm701466p

A series of 5-alkylindolobenzazepin-7-ones was synthesized by Suzuki coupling between 3-iodoindole-2-carboxylates and the appropriate α-alkylbenzylamino α-boronic acids followed by cyclization to the lactam. Derivatives having a linear alkyl chain at C5 were found to be highly cytotoxic to KB cells with IC₅₀ values in the 30-80 nM range. These compounds also inhibited the polymerization of tubulin with IC₅₀'s of 1-2 μM. Compound 4f ((S)-5-ethyl) showed comparable antiproliferative activities (IC₅₀'s of 30-70 nM) in a variety of cancer cell lines, cell growth being arrested at the G2/M phase. Compound 4f induced apoptosis in a dose-dependent manner in three different cancer cell lines and was shown to affect cell morphology in a manner consistent with its inhibitory action on tubulin polymerization. Using the experimental model of glioma grafted on the chick chorio-allantoic membrane, local treatment with compound 4f markedly reduced tumor progression.

Full text of DOI:10.1021/jm701466p

3414
J. Med. Chem. 2008, 51, 3414–3421
New C5-Alkylated Indolobenzazepinones Acting as Inhibitors of Tubulin Polymerization:
Cytotoxic and Antitumor Activities
Laurent Keller, Ste´phane Beaumont, Jian-Miao Liu, Sylviane Thoret, Je´roˆme S. Bignon, Joanna Wdzieczak-Bakala,
Philippe Dauban,* and Robert H. Dodd*
Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, AVenue de la Terrasse, 91198 Gif-sur-YVette, France
ReceiVed NoVember 21, 2007
A series of 5-alkylindolobenzazepin-7-ones was synthesized by Suzuki coupling between 3-iodoindole-2-
carboxylates and the appropriate R-alkylbenzylamino o-boronic acids followed by cyclization to the lactam.
Derivatives having a linear alkyl chain at C5 were found to be highly cytotoxic to KB cells with IC50 values
in the 30-80 nM range. These compounds also inhibited the polymerization of tubulin with IC50’s of 1-2
µM. Compound 4f ((S)-5-ethyl) showed comparable antiproliferative activities (IC50’s of 30-70 nM) in a
variety of cancer cell lines, cell growth being arrested at the G2/M phase. Compound 4f induced apoptosis
in a dose-dependent manner in three different cancer cell lines and was shown to affect cell morphology in
a manner consistent with its inhibitory action on tubulin polymerization. Using the experimental model of
glioma grafted on the chick chorio-allantoic membrane, local treatment with compound 4f markedly reduced
tumor progression.
Introduction
and antitumor properties.11b These include synthetic N-acetyl-
colchinol (NAC,a 2a) and its water-soluble phosphate prodrug
form 2b (ZD6126),12 which are structurally simpler analogues
of colchicine, their recently described heterocyclic analogues
2c,13 as well as the natural products combretastatin A-4 (3) and
podophyllotoxin.14 Interestingly, while a wide variety of
analogues of these compounds can be accessed relatively easily,
no compounds binding to the colchicine site have yet found
their way into the clinic as anticancer agents.5
The indole nucleus is a structural component of a wide variety
of antimitotic compounds.15 We thus decided to investigate the
effect of replacing one of the phenyl rings of the antimitotic
compounds of type 2 by an indole moiety by synthesizing
5-substituted indolobenzazepinones of general structure 4. We
describe herein the preparation of these compounds and report
their potent cytotoxic and antimitotic properties, which allow
us to conclude that 5-alkylindolobenzazepinones represent a new
class of antimitotic agents demonstrating antitumor activity.
Microtubules are important for the formation of the mitotic
spindle during the process of mitosis. They have a major role
to play in maintaining the growth, division, and functioning of
both normal and cancer cells as well as being implicated in
motility, shape maintenance, and intracellular transport.1 Mi-
crotubules are formed by polymerization of two closely related
heterodimers composed of R- and ꢀ-tubulin subunits. Interfer-
ence with the dynamic equilibrium between the assembly of
tubulin into microtubules or, inversely, the depolymerization
of microtubules into tubulin leads to arrested cell division and
eventually to apoptosis. Compounds that can inhibit either of
these important processes are thus referred to as spindle poisons
or antimitotics.2,3
The search for selective inhibitors of tubulin assembly or
disassembly has led to the development of some of the most
clinically useful antitumor drugs currently in use.4,5 In the first
group are the naturally occurring Vinca alkaloids vincristin and
vinblastin as well as their synthetic analogues (e.g., navelbine).6a
The taxoids (taxol, taxotere), which are inhibitors of microtubule
depolymerization, are widely used for the treatment of breast,
ovarian, and nonsmall cell lung carcinomas.6b Epothilone6c and
discodermolide6d are also two potent microtubule-stabilizing
agents that have undergone intensive clinical evaluation.
However, these complex molecules are generally difficult to
synthesize, are toxic, and become rapidly prone to resistance
phenomena.2–5
Chemistry
The general strategy for the synthesis of the indolobenzaze-
pinones of type 4 consisted of Suzuki coupling of the o-boronic
acid of an appropriately substituted benzylamine with a 3-io-
doindole-2-carboxylate, followed by intramolecular cyclization
after amine deprotection. Thus, the aminobenzyl o-boronic acids
7a-i were first prepared as shown in Scheme 1. Benzylamine
(5a) and the commercially available racemic or optically pure
R-methyl and R-ethyl benzylamines (5b-5f) were protected as
their N-Boc derivatives 6a-6f. The noncommercial, racemic
R-n-propyl-, R-n-butyl-, and R-i-propylbenzylamines 5g-5i,
respectively, were prepared by Leuckart reductive amination16
of the precursor ketones, which were subsequently N-Boc
protected to give 6g-6i. These N-Boc-benzylamines were then
ortho-lithiated using t-butyllithium in THF and reacted with
The antitumor action of colchicine (1, Chart 1), a natural
product isolated from Colchicum autumnale L., has been known
for a long time.7 This compound, which acts as an inhibitor of
tubulin polymerization,8 binds to ꢀ-tubulin at the interface with
R-tubulin, a site different from that of the Vinca alkaloids.9,10
However, its narrow therapeutic index precludes its clinical
use.11 A number of other compounds are known to bind to the
colchicine site of tubulin and consequently display cytotoxic
a Abbreviations: CAM, chorio-allantoic membrane; DMEM, Dulbecco
minimal essential medium; EGTA, [ethylene bis(oxyethylenenitrilo)]tet-
raacetic acid; EMEM, Eagle’s minimal essential medium; FCS, fetal calf
serum; GTP, guanosine triphosphate; MES, 2-(N-morpholino)ethanesulfonic
acid; NAC, N-acetylcolchinol; PBS, phosphate buffered saline.
* To whom correspondence should be addressed. Phone: 33-1-69824560
(P.D.); 33-1-69824594 (R.H.D.). Fax: 33-1-69077247 (P.D.); 33-1-69077247
(R.H.D.). E-mail: philippe.dauban@icsn.cnrs-gif.fr (P.D.); robert.dodd@
icsn.cnrs-gif.fr (R.H.D.).
10.1021/jm701466p CCC: $40.75
2008 American Chemical Society
Published on Web 05/27/2008

C5-Alkylated Indolobenzazepinones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3415
Chart 1. Structures of Various Known Antimitotic Agents and of the 5-Alkyl Indolobenzazepinones
Scheme 1a
a Reagents and conditions (for reaction (a)): (a) HCO2H, HCONH2, 180 °C, 16 h; (b) aq EtOH, HCl, reflux, 8 h. Reagents and conditions (for reaction
(b)): (a) Boc2O, Et3N, DCM, room temperature, 24 h; (b) t-BuLi (2.8 eq), THF, -78 °C to -20 °C, 2.5 h; (c) B(OMe)3 (5 eq), -20 °C to room temperature,
2 h; (d) 5% aq HCl, 0 °C.
trimethylborate to give the corresponding o-boronates.17 Hy-
drolysis of these products with aqueous HCl afforded the desired
boronic acids 7a-7i, generally mixed with unreacted starting
material, which were then used for the subsequent coupling
reaction without purification.
of the carbonyl group of 4b with lithium aluminum hydride/
aluminum chloride afforded the benzazepine derivative 12.
The effect on activity of eliminating one or both of the NH
groups of the indolobenzazepinones was evaluated by preparing
first the di-N-methylated derivative 13 via reaction of 4b with
sodium hydride and excess methyl iodide. Selective methylation
of the indole nitrogen to give 14 was achieved using methyl
iodide under phase transfer conditions.
The required 3-iodoindole-2-carboxylates 9 were prepared as
shown in Scheme 2. Commercial indole 2-carboxylic acids
8a-8c were first transformed into the corresponding ethyl esters.
These were reacted with iodine in DMF in the presence of
potassium hydroxide, affording the 3-iodoindole-2-carboxylate
derivatives in good yields.18 The indole nitrogen of each
compound was then protected with a benzenesulfonyl group to
give 9a-9c. The latter were subjected to a palladium-catalyzed
Suzuki coupling reaction19 with the benzylboronic acids 7a-7i
to give the corresponding C3 coupled products 10a-10k in
yields ranging from 42 to 81%. The benzylamino group of
compounds 10 was then deprotected with trifluoroacetic acid
in dichloromethane. Treatment of the resulting amines with
sodium in ethanol led to cyclization with concomitant depro-
tection of the indole nitrogen, providing the desired indoloben-
zazepinones 4a-4k.
Results and Discussion
In Vitro Cell Growth Inhibition. The cytotoxic properties
of the various indolobenzazepinone derivatives synthesized
(4a-4k, 11-14) were first evaluated on KB cells (Table 1).
While the completely unsubstituted compound 4a displayed only
modest activity (IC50 ) 0.550 µM),20 introduction of a racemic
methyl group at C5 (compound 4b) led to a doubling of
cytotoxicity (IC50 ) 0.280 µM). When the two 5-methyl
enantiomers were tested separately, it was found that the (S)-
isomer 4d was considerably more active than the (R)-isomer
4c (IC50 ) 0.080 and 0.340 µM, respectively). Replacement of
the methyl group by an ethyl group also resulted in higher
cytotoxicity, although this time there was not a considerable
difference between the (R)- and the (S)-isomers 4e (IC50 ) 0.035
µM) and 4f (IC50 ) 0.037 µM), respectively. Elongation of the
alkyl chain at C5 had little incidence on activity. Thus, both
To study the importance of the carbonyl group of the
benzazepinone ring for the activity of these compounds,
compound 4b was transformed into its thiocarboxamide ana-
logue 11 using Lawesson’s reagent (Scheme 3), while reduction

3416 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Keller et al.
Scheme 2a
Table 1. Cytotoxic Effects of Compounds 4a-4k and Derivatives with
Respect to KB cellsa and Inhibition of Tubulin Polymerization by
Selected Compounds
cytotoxicity
R1 R2 R3 R4 IC50 [µM]b IC50 [µM]d
ITPc
compd
R
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
Me
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
S
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.550
0.280
0.340
0.080
0.035
0.037
0.032
0.035
0.280
3.50
0.500
0.360
12.0
28.0
7.0
nde
nd
nd
1.9
1.8
1.9
2.0
1.0
1.7
51
3.6
nd
nd
nd
nd
(R)-Me
(S)-Me
(R)-Et
(S)-Et
n-Pr
n-Bu
i-Pr
(S)-Me OMe
4j
4k
11
12
13
14
Et
F
Me
Me
Me
Me
H
H
H
H
a Reagents and conditions: (a) EtOH, cat. H2SO4, reflux, 16 h; (b) KOH,
I2, DMF, room temperature, 45 min; (c) PhSO2Cl, NaH, THF, room
temperature, 16 h; (d) 7a-7i (1.2 eq), Pd(PPh3)4 (10 mol%), Na2CO3 (2
eq), toluene-EtOH (1:1), reflux, 20 h; (e) TFA, DCM, room temperature,
16 h; (f) Na/EtOH, reflux, 10 h.
2H
O
O
Me Me
Me
H
colchicine (1)
NAC (2a)
0.020
0.075
2.2
3.0
a KB ) cervical carcinoma cells. b IC50 is the concentration of compound
inducing 50% cell growth inhibition after 72 h incubation. c Inhibition of
tubulin polymerization. d IC50 is the concentration of compound required
to inhibit 50% of the rate of microtubule assembly; values representative
of three experiments. e Not determined.
Scheme 3a
(13) or only the indole nitrogen (14) led to severe loss of activity
(IC50 ) 28 and 7 µM, respectively).
While compounds 4e-4h all present essentially the same
cytotoxic activities, compound 4f was selected for further study
because of its availability in enantiomerically pure form from
commercially available 5f. The cytotoxic activity of 4f in a
variety of cancer cell lines was first determined and in some
cases compared to that of colchicine (1) and NAC (2a) (Table
2). Thus, the cytotoxicity of 4f in all cell lines was of the same
order of magnitude as in KB cells, IC50’s ranging from 0.030
µM in MDA-MB231 cells to 0.070 µM in MDA-MB435 and
K562 cells, thus clearly indicating an antiproliferative activity
regardless of the origin of the tumor cells. Compound 4f was
as active as colchicine in A549 and HCT116 cells but about
twice less active in B16F10 and MDA-MB435 cells. Inversely,
4f was over twice as cytotoxic to MDA-MB231 than colchicine.
On the other hand, 4f was 2-fold more active than NAC in
MDA-MB435 and HCT116 cells, 5-fold more active in A549
cells and over 20 times more active in MDA-MB231 cells, while
the cytotoxicities of these two compounds were identical in
B16F10 cells (IC50 ≈ 0.070 µM).
a Reagents and conditions: (a) Lawesson’s reagent, toluene, reflux, 30
min; (b) AlCl3 (0.5 eq), LiAlH4 (1 eq), THF, 4 h at 60 °C then 12 h at
reflux; (c) NaH (4 eq), CH3I (6 eq), THF, room temperature, 2 h; (d) CH3I
(10 eq), cat. TBAB, toluene, H2O, room temperature, 2 h.
the racemic n-propyl (4g) and n-butyl (4h) homologues were
as cytotoxic (IC50 ) 0.032 and 0.035 µM, respectively) as the
C5 ethyl compounds 4e,f. These activities are comparable to
those of colchicine (1, IC50 ) 0.020 µM) and NAC (2a, IC50
)
0.075 µM) in the same cell line. However, a more sterically
demanding alkyl group at this position was less active, as
demonstrated by the isopropyl analogue (4i) (IC50 ) 0.280 µM).
Small substituents at the C11 position of the more active
indolobenzazepinone derivatives did not enhance the biological
activity. Thus, both the 5-methyl-11-methoxy and the 5-ethyl-
11-fluoro analogues (4j and 4k, respectively) were considerably
less cytotoxic (IC50 ) 3.50 and 0.500 µM) than their C11
nonsubstituted counterparts 4d and 4e,f (IC50 ) 0.080 and 0.035
µM). With regard to the carboxamide function, its replacement
by a thiocarboxamide had a small detrimental effect on activity
(11, IC50 ) 0.360 µM compared to 4b, IC50 ) 0.280 µM), while
its reduction to an amine practically abolished the cytotoxicity
(12, IC50 ) 12 µM). Finally, methylation of both nitrogen atoms
Effect on Cell Cycle Progression. The effect of compound
4f on the cell cycle of K562, MCF7, and MDA-MB231 cells
was then investigated by flow cytometry at various concentra-
tions (Table 3). After 24 h treatment with 0.05 µM of 4f, a net
increase in the number of cells arrested at the G2/M growth
phase was observed. Increasing the concentration of 4f to 0.1
µM led to essentially the same result in K562 and MCF7 cells
(20.3% and 62.6% in G2/M phase, respectively), while practi-
cally all MDA-MB231 cells were arrested in the G2/M phase
at this concentration. A concentration of 0.5 µM of compound
4f was required to arrest 44.7% of K562 cells in the G2/M phase.
In K562 cells, the percentage of cells arrested in the G2/M phase
tended to increase with concentration of 4f (0.05, 0.1, 0.5 µM)
regardless of length of treatment (6, 16, 24 h) (see Supporting

C5-Alkylated Indolobenzazepinones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3417
Table 2. Antiproliferative Activities of 4f, 1, and 2a against Various Cancer Cell Lines after 72 h Treatment (IC50’s, µM)a
compound
4f
colchicine (1)
NAC (2a)
B16F10
A549
MDA-MB435
MDA-MB231
MCF7
L1210
K562
HT29
HCT116
0.068
0.029
0.072
0.050
0.045
0.250
0.070
0.040
0.140
0.030
0.070
0.700
0.040
ndb
nd
0.034
nd
nd
0.070
nd
nd
0.042
nd
nd
0.042
0.040
0.100
a IC50 is the concentration of test compound inducing 50% cell growth inhibition after 72 h incubation; cell lines: B16F10 ) skin melanoma, A549 )
human nonsmall cell lung cancer, MDA-MB435, MDA-MB231, MCF7 ) breast cancer, L1210, K562 ) leukemia, HT29, HCT116 ) human colon cancer.
b Not determined.
Table 3. Percentage of K562, MCF7, and MDA-MB231 Cancer Cells
in the G2/M Phase after 24 h of Treatment with Compound 4fa
concentration
of 4f (µM)
K562
(% G2/M)
MCF7
(% G2/M)
MDA-MB231
(% G2/M)
control
0.05
0.1
3.9
24.9
20.3
44.7
10.7
65.6
62.6
ndb
25.9
44.5
∼100
nd
0.5
a Data are representative of three independent experiments. b Not
determined.
Information). In all cells treated with 4f for 24 h, a subdiploid
DNA content was observed (data not shown), indicating that
cells are undergoing apoptosis, probably as a result of the cell
cycle being arrested in the G2/M phase.21
Effect on Apoptosis. The ability of compound 4f to induce
apoptosis was further characterized by a specific apoptosis assay.
Cleavage of pro-caspases to active caspases is one of the
hallmarks of apoptosis. K562, HCT116, and MDA-MB231 cell
lines were thus treated with 0.01, 0.1, and 1 µM of compound
4f and caspase 3 and 7 activities were evaluated using the
standard caspase cleavage assays (Figure 1). It was observed
that, after 24 h of treatment, 4f induced apoptosis in all three
investigated cell lines. In particular, a 6-fold increase in
apoptosis occurred in K562 leukemia cells previously described
as being resistant to apoptosis induction by a variety of agents
including diphtheria toxin, camptothecin, cytarabine, etoposide,
paclitaxel, staurosporine, and antifas antibodies.22–27 These
results show that treatment of cancer cells with compound 4f
activates caspases leading to cellular apoptosis.
In Vitro Tubulin Polymerization Inhibition. The structural
resemblance of the indolobenzazepinones with colchicine (1)
and NAC (2a) as well as the observation that 4f arrests the cell
cycle at the G2/M phase21 encouraged us to investigate the
ability of the more cytotoxic compounds of type 4 to inhibit
tubulin polymerization. Indeed, the high cytotoxicities of
compounds 4d-4h are well correlated with their ability to
effectively inhibit tubulin polymerization, their IC50’s all being
in the 1-2 µM range and thus comparable to the inhibitory
activity of colchicine and NAC (IC50 ) 2.2 and 3.0 µM,
respectively, Table 1). On the other hand, compound 4i
displayed cytotoxicity, which was 10-fold less than the most
active compounds yet inhibited tubulin polymerization with the
same order of potency (IC50 ) 1.7 µM). This may reflect the
fact that compound 4i, while effectively binding to tubulin in
vitro, has difficulty penetrating into cells, a prerequisite for
cytotoxicity. The methoxy derivative 4j displays very weak
cytotoxicity with respect to KB cells (IC50 ) 3.5 µM) and a
correspondingly high IC50 for inhibition of tubulin polymeri-
zation (51 µM). This is compatible with tubulin being the
principal target of these C5-alkylated indolobenzazepinones.
Interestingly, tubulin polymerization inhibition and, to a lesser
extent, cytotoxicity, are restored by replacing the methoxy group
of 4j by a fluoride (4k, IC50 ) 3.6 and 0.500 µM with respect
to tubulin activity and cytotoxicity, respectively).
Figure 1. Induction of apoptosis in three different cancer cell lines
by 24 h treatment with 4f as a function of concentration.
disruption can induce G2/M arrest and apoptosis, triple fluo-
rescence labeling was employed to analyze cell morphology.
As shown in Figure 2A (60× magnification), 24 h after
treatment with 4f at 0.050 µM, MDA-MB231 cells differed in
size and shape, the cell mass became smaller, and the cell body
was shrunken. Some of the exposed cells were binucleated,
multinucleated, or micronucleated, indicating dividing failure
and mitotic catastrophe (Figure 2B, 100× magnification).
Notably, the cells had altered fibrillar array of actin filaments,
and it was clearly seen that 4f caused abnormal cellular
microtubule organization and arrangement in these cells, further
evidence that the cytotoxic effects of this compound are the
result of its interaction with tubulin. Similar observations have
been made by others using tubulin-disrupting agents.28,29
Antitumor Effect. Finally, the effect of compound 4f on
tumor growth was determined in vivo and compared to the
effects of colchicine and NAC. Human glioma growth was
assessed using an avian embryo model. The experimental glioma
grafted on the chick chorio-allantoic membrane (CAM) repli-
cates characteristics of the human disease and allows rapid
testing of new anticancer drugs.30 Local treatment of size-
matched glioma from day 3 to day 6 with single daily doses
(10 µM) of 4f markedly inhibited tumor progression (Figure
3). After 4 days of treatment, tumor volume decreased by 76%
while the control tumor volume increased by 20%. By com-
parison, colchicine (10 µM) and NAC (100 µM) decreased
tumor volume by only 50% and 42%, respectively, after 4 days
of treatment.
Conclusion
We have shown that 5-alkylindolobenzazepin-7-ones of
general structure 4 are potent antiproliferative agents across a
wide variety of cancer cell lines. The observed cytotoxic effects
are most probably due to interaction with tubulin. This is
indicated by equipotency of 4f with the antimitotics colchicine
Effect on Cell Morphology. Because microtubules as well
as microfilaments are essential for cell division and their

3418 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Keller et al.
Figure 3. In vivo effect of compound 4f on the size of a human glioma
(U87) tumor transplanted in the chick chorio-allantoic membrane
(CAM). (A) Microscopic images of tumor size regression produced
by treatment with 10 µM of compound 4f 3 days (D3), 5 days (D5),
and 7 days (D7) after tumor transplant (bottom) compared to control
(top) which received no treatment. (B) Percentage of tumor regression
caused by local treatment with compound 4f (10 µM, 9), colchicine
(10 µM, 2), and NAC (100 µM, b) compared to control ([).
Spectrum BX FT-IR spectrometer. Optical rotations were deter-
mined with a JASCO P-1010 polarimeter. Proton (1H) and carbon
(13C) NMR spectra were recorded on Bruker spectrometers: AC
250 (250 MHz), Aspect 3000 (300 MHz), Avance 300 NMR or
500 NMR (respectively 300 and 500 MHz). Chemical shifts (δ)
are reported in parts per million (ppm) with reference to tetram-
ethylsilane (TMS) as internal standard. NMR experiments were
carried out in deuterochloroform (CDCl3) or in deuterobenzene
(C6D6). The following abbreviations are used for the proton spectra
multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; qu, quintet;
m, multiplet; td, triplet of doublets; tt, triplet of triplets; sept, septet.
Coupling constants (J) are reported in Hertz (Hz). Mass spectra
were obtained either with an AEI MS-9 instrument using electron
spray (ESI-MS) or with a MALDI-TOF instrument for high
resolution mass spectra (HREIMS). Thin-layer chromatography was
performed on silica gel 60 plates with a fluorescent indicator and
visualized under a UVP Mineralight UVGL-58 lamp (254 nm) and
with a 7% solution of phosphomolybdic acid in ethanol. Flash
chromatography was performed using silica gel 60 (40-63 µm,
230-400 mesh ASTM) at medium pressure (200 mbar). All
solvents were distilled and stored over 4 Å molecular sieves before
use. All reagents were obtained from commercial suppliers unless
otherwise stated. Organic extracts were, in general, dried over
magnesium sulfate (MgSO4) or sodium sulfate (Na2SO4). Elemental
analyses were performed at the ICSN, CNRS, Gif-sur-Yvette,
France.
Figure 2. Effect of compound 4f (50 nM) on the morphology of MDA-
MB231 cells after 24 h of treatment. (1) tubulin, (2) actin, (3) nucleus,
(4) merged image; (A) 60× magnification; (B) merged image of MDA-
MB231 cells (100× magnification).
and NAC in inhibiting tubulin polymerization, by the interrup-
tion of cancer cell growth at the G2/M stage in the presence of
4f, and by the morphological changes seen in cancer cells treated
with this compound typical of antimitotic agents. The ability
of 4f to promote apoptosis in the cancer cells studied was also
clearly demonstrated. Finally, these in vitro observations were
confirmed in vivo whereby compound 4f, used in a CAM model
of tumor growth at concentrations inhibiting tubulin polymer-
ization, was significantly more effective than colchicine and
NAC in causing tumor volume regression after only several days
of treatment. Further structure-activity studies in this series
are in progress as are in vivo studies in xenografted mice.
Experimental Section
Chemistry. Melting points, measured in capillary tubes and
recorded using a Bu¨chi B-540 melting point apparatus, are
uncorrected. Infrared spectra were recorded on a Perkin-Elmer
Complete procedures for the preparation of compounds 4f,
11-14 are given below. For general procedures and characterization
of the other compounds, see Supporting Information.

C5-Alkylated Indolobenzazepinones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3419
(S)-N-(tert-Butyloxycarbonyl)-r-ethylbenzylamine (6f). A so-
lution of di-tert-butyl dicarbonate (4.58 g, 21 mmol) in dry DCM
(11 mL) was added under argon at 0 °C to a solution of (S)-R-
ethylbenzylamine 5f (2.32 g, 15 mmol) and triethylamine (2.09 mL,
15 mmol) in dry DCM (33 mL). The reaction mixture was stirred
at room temperature for 24 h. The solvent was removed in vacuo,
and the residue was dissolved with DCM (60 mL) and water (40
mL). The aqueous layer was extracted with DCM (3 × 50 mL).
The combined organic extracts were washed with water (25 mL),
dried (MgSO4), filtered, and concentrated. The resulting white solid
was purified by flash chromatography on silica gel (heptane/AcOEt
9/1) to give compound 6f in 73% yield as a white solid; mp 68 °C
(Et2O). 1H NMR (250 MHz, CDCl3) δ: 7.33 (m, 5H), 4.92 (m,
1H), 4.65 (m, 1H), 1.83 (m, 2H), 1.47 (s, 9H), 0.95 (t, J ) 7.5 Hz,
3H). 13C NMR (75 MHz, CDCl3) δ: 155.3, 142.9, 128.4, 127.0,
126.3, 79.3, 56.3, 29.8, 28.3, 10.6. FTIR (ν/cm-1) 3384, 1682, 1517.
[R]D ) -44.6 (CHCl3, c ) 6.06). Anal. (C14H21NO2) C, H, N.
(S)-N-(tert-Butyloxycarbonyl)-r-ethylbenzylamino-2-boronic acid
(7f). tert-Butyllithium (4.94 mL, 8.4 mmol, 1.7 M in pentane) was
added under argon at -78 °C to a solution of 6f (3 mmol) in dry
THF (15 mL). The reaction mixture was allowed to warm to -20
°C for 2.5 h. Trimethylborate (2 mL, 15 mmol) was added at -20
°C, and the reaction was allowed to warm to rt for 2 h. A 5%
aqueous solution of HCl was added at 0 °C with vigorous stirring
until a pH ) 5-6 was attained (≈ 3 mL). The aqueous layer was
extracted with DCM (3 × 25 mL). The combined organic extracts
were washed with brine (20 mL), dried (MgSO4), filtered, and
evaporated in vacuo to give an approximately 1:1 mixture of boronic
acid 7f and starting material 6f (as estimated by NMR). The crude
boronic acid was used in the following step without further
purification.
added to a solution of iodoindole 9a (0.260 g, 0.57 mmol), o-boronic
acid 7f (0.207 g, 0.74 mmol), and tetrakis(triphenylphosphine)-
palladium(0) (0.062 g, 0.04 mmol) in toluene/EtOH 1/1 (10 mL).
The reaction mixture was stirred at reflux for 20 h. After cooling,
ice was added and the mixture was extracted with AcOEt (3 × 20
mL). The combined organic extracts were washed with water (10
mL), dried (MgSO4), filtered, and evaporated. The resulting oil was
purified by flash chromatography on silica gel (heptane/AcOEt 95/5
then 75/25) to give compound 10f as a mixture of atropoisomers
1
in 67% yield; white foam. H NMR (250 MHz, CDCl3) δ: 8.10
(m, 3H), 7.74 (d, J ) 7.4 Hz, 1H), 7.63-7.10 (m, 8H), 4.85 (m,
1H), 4.39 (m, 1H), 4.22 (q, J ) 7.1 Hz, 2H), 2.25 (m, 1H), 1.93
(m, 1H), 1.57 (s, 9H), 1.09 (t, J ) 7.1 Hz, 3H), 0.51 (t, J ) 7.4
Hz, 3H). 13C NMR (75 MHz, CDCl3) δ: 162.1, 152.8, 141.9, 137.7,
135.8, 133.9, 131.4, 130.5, 129.6, 129.5, 129.0, 127.7, 127.4, 127.3,
126.8, 125.3, 124.5, 124.3, 122.2, 114.8, 79.4, 61.9, 53.8, 30.2,
28.2, 13.7, 10.2. FTIR (ν/cm-1) 3347, 1724, 1681, 1173. HRMS
(ESI+) calcd for [C31H34N2O6SNa]+ 585.2035, found 585.2040.
Anal. (C31H34N2O6S·0.85C2H5OH·0.85H2O) C, H, N.
(S)-5-Ethyl-5,8-dihydroindolo[2,3-d][2]benzazepin-7(6H)-one (4f).
Trifluoroacetic acid (0.18 mL, 2.3 mmol) was added at 0 °C to a
solution of N-Boc protected derivatives 10f (0.186 g, 0.33 mmol)
in dry DCM (2.4 mL). The reaction mixture was stirred at rt
overnight. Solvent and excess TFA were evaporated in vacuo and
the residue was dissolved in DCM and made basic with a 1 M
aqueous solution of NaOH. The aqueous layer was extracted with
DCM (3 × 10 mL). The combined organic extracts were washed
with brine (5 mL), dried (MgSO4), filtered, and evaporated in vacuo
to give the free amine. This crude product (74 mg, 0.16 mmol)
was dissolved in absolute ethanol (2 mL) and a solution of sodium
(29 mg, 1.3 mmol) in absolute ethanol (2 mL) was added. The
reaction mixture was stirred at reflux for 10 h. Silica was added
and the solvent was evaporated in vacuo. The resulting residue was
purified by flash chromatography on silica gel (heptane/AcOEt 9/1
then 3/7) to give lactams 4f in 72% yield as a white solid; mp 157
Ethyl 1-(Benzenesulfonyl)-1H-3-iodoindole-2-carboxylate (9a).
Sulfuric acid (2 mL) was added to a solution of indole-2-carboxylic
acid 8a (3.22 g, 20 mmol) in absolute ethanol (60 mL). The reaction
mixture was refluxed overnight. After cooling to rt, the solvent was
removed under vacuum, the residue was dissolved in DCM (50
mL), and the resulting solution was washed with a 1 M aqueous
solution of NaHCO3 (3 × 20 mL) and water (2 × 20 mL), dried
(MgSO4), filtered, and evaporated in vacuo to give the correspond-
ing ethyl ester as a solid. The ethyl ester (3.59 g, 19.0 mmol) was
dissolved in DMF (11 mL) and potassium hydroxide (4.05 g, 72.4
mmol) was added portionwise. The reaction mixture was stirred at
rt for 15 min, and a solution of iodine (4.84 g, 19.0 mmol) in DMF
(11 mL) was added. The reaction mixture was stirred at rt for a
further 45 min and then poured into a mixture of a 25% aqueous
solution of NaHSO3 (8 mL), 33% aqueous solution of NH4OH (16
mL), and water (320 mL). The resulting precipitate was filtered
and dried in vacuo to give a yellow solid, which was dissolved in
hot EtOH (60 mL) and crystallized at 0 °C to give the iodoindole
as needles. A solution of the iodoindole (14.8 mmol) in THF (70
mL) was added at 0 °C under argon to a suspension of sodium
hydride (0.89 g, 22.3 mmol, 60% in oil) in dry THF (70 mL). The
reaction mixture was stirred for 45 min, and benzenesulfonyl
chloride (6.0 mL, 29.8 mmol) was added. The reaction mixture
was warmed to rt and left overnight. The solvent was evaporated
in vacuo, and the residue was dissolved with DCM (100 mL) and
water (20 mL). The aqueous layer was extracted with DCM (3 ×
20 mL). The combined organic extracts were washed with water
(40 mL) and a 1 M aqueous solution of NaHCO3 (2 × 20 mL),
dried (MgSO4), filtered, and evaporated in vacuo. The resulting
brown oil was dissolved in hot hexane/Et2O (1/1) and crystallized
at 0 °C to give compound 9a in 64% yield as a white solid; mp
1
°C (heptane/AcOEt). H NMR (250 MHz, CDCl3) δ: 11.61 (br s,
1H), 8.07 (m, 2H), 7.56 (m, 4H), 7.33 (m, 4H), 4.12 (m, 1H), 2.20
(m, 2H), 1.15 (m, 3H). 13C NMR (75 MHz, CDCl3) δ: 164.9, 139.9,
138.4, 137.1, 133.9, 128.7, 128.5, 127.7, 126.9, 125.1, 121.5, 123.8,
120.9, 119.0, 112.8, 54.7, 30.3, 11.6. FTIR (ν/cm-1) 3179, 1635.
HRMS (ESI) calcd for [C18H16N2ONa] 299.1160, found 299.1175.
[R]D ) -77 (CHCl3, c ) 0.09). Anal. (C18H16N2O·0.2CH3CO2-
C2H5 ·0.2C7H16) C, H, N.
(R,S)-5-Methyl-5,8-dihydroindolo[2,3-d][2]benzazepin-7(6H)-
thione (11). Lawesson’s reagent (15 mg, 0.038 mmol) was added
under argon to a suspension of lactam 4b (10 mg, 0.038 mmol) in
dry toluene (0.5 mL). The reaction mixture was stirred at reflux
for 30 min. Silica was added and solvent was evaporated in vacuo.
The residue was purified by flash chromatography on silica gel
(CH2Cl2/AcOEt 98/2) to give thiolactam 11 as a pale-yellow solid
(7 mg, 65% yield). 1H NMR (250 MHz, CDCl3) δ: 9.50 (br s,
1H), 8.06 (m, 1H), 7.75 (br s, 1H), 7.53-7.12 (m, 7H), 4.40 (m,
1H), 1.88 (m, 3H). 13C NMR (75 MHz, CDCl3) δ: 185.2, 137.6,
137.2, 133.4, 133.2, 128.6, 128.2, 127.9, 126.2, 125.9, 123.5, 122.5,
121.6, 112.3, 76.2, 52.7, 16.7. FTIR (ν/cm-1) 3416, 1556. HRMS
(ESI+) calcd for [C17H15N2S]+ 279.0956, found 279.0968.
(R,S)-5-Methyl-5,6,7,8-tetrahydro-6H-benzo[5,6]azepino[3,4-
b]indole (12). A solution of aluminum chloride (45 mg, 0.34 mmol)
in dry THF (1 mL) was added under argon to a suspension of
lithium aluminum hydride (26 mg, 0.68 mmol) in dry THF (1 mL).
The reaction mixture was stirred for 30 min at rt and a solution of
lactam 4b (90 mg, 0.34 mmol) in dry THF (1 mL) was added. The
reaction mixture was stirred for 4 h at 60 °C. After cooling,
additional lithium aluminum hydride (26 mg, 0.68 mmol) was added
and the mixture was stirred overnight at reflux. After cooling, a
THF/H2O (9/1) mixture was slowly added until gas evolution ceased
followed by addition of a 1 M aqueous solution of NaOH until
basic pH was attained. The mixture was extracted with AcOEt (3
× 10 mL). The combined organic extracts were dried (MgSO4),
filtered, and evaporated in vacuo. The resulting oil was purified by
flash chromatography on silica gel (AcOEt/MeOH 100/0 to 80/20)
1
139 °C (hexane/Et2O). H NMR (250 MHz, CDCl3) δ: 8.00 (d, J
) 7.5 Hz, 3H), 7.60-7.30 (m, 6H), 4.54 (q, J ) 7.1 Hz, 2H), 1.48
(t, J ) 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ: 161.7, 137.3,
135.5, 134.2, 133.4, 131.4, 129.1, 127.4, 127.3, 124.8, 123.1, 114.7,
73.5, 62.8, 14.0. FTIR (ν/cm-1) 3065, 1729, 1192, 726. Anal.
(C17H14INO4S) C, H, N.
(S)-Ethyl 1-(Benzenesulfonyl)-3-{2-[1-(tert-butyloxycarbonyl-
amino)propyl]phenyl}-1H-indole-2-carboxylate (10f). Under argon,
a 1.5 M aqueous solution of Na2CO3 (0.75 mL, 1.1 mmol) was

3420 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Keller et al.
1
to give benzazepine 12 as a white solid (70 mg, 83% yield). H
NMR (250 MHz, CDCl3) δ: 8.35 (br s, 1H), 7.93 (m, 2H), 7.43
(m, 3H), 7.18 (m, 3H), 4.43 (d, J ) 16.5 Hz, 1H), 4.24 (d, J )
16.5 Hz, 1H), 4.06 (q, J ) 6.6 Hz, 1H), 1.59 (d, J ) 6.6 Hz, 3H).
13C NMR (75 MHz, CDCl3) δ: 142.1, 136.1, 135.9, 134.7, 128.1,
127.2, 126.8, 125.2, 125.0, 122.1, 120.2, 119.5, 113.0, 111.0, 53.2,
45.5, 19.0. FTIR (ν/cm-1) 3395, 739. MS (ESI+): 249 [M + H+].
HRMS (ESI+) calcd for [C17H17N2]+ 249.1392, found 249.1422.
by flow cytometry on a FC500 flow cytometer (Beckman-Coulter,
France) as described previously.31
Apoptosis Assay. Apoptosis was measured by the Apo-one
homogeneous caspase-3/7 assay (Promega Co, WI) according to
the manufacturer’s recommendations. Briefly, cells were subcultured
on a 96-well plate with 5 × 104 cells/well in 100 µL medium.
After 24 h of incubation, the medium in the 96-well plate was
discarded and replaced with medium containing different concentra-
tions of compound 4f (1, 0.1, and 0.01 µM) or 0.1% DMSO (as
negative control). The treated cells were incubated for 24 h, each
well then received 100 µL of a mixture of caspase substrate and
Apo-one caspase 3/7 buffer. After 1 h of incubation, the fluores-
cence of sample was measured using a Victor microtiter plate
fluorimeter (Perkin-Elmer, USA) at 527 nm.
Immunocytochemistry. MDA-MB231 cells cultured on a
Laboratory-Tek chamber slide (VWR International, Strasbourg,
France) were treated with 50 nM of compound 4f for 24 h. At the
end of treatment, cells were fixed with 3.7% formaldehyde and
then permeabilized with 0.1% Triton X-100 in PBS for 3 min at
room temperature. The cells were first incubated for 1 h in a solution
of PBS containing 1% BSA and 10% goat serum to block
nonspecific antibody binding and next for 12 h at 4 °C with the
mouse anti-ꢀ tubulin antibody (1:100) (Zymed, Invitrogen, Cergy
Pontoise, France). Cells incubated with preimmune mouse serum
instead of primary antibody were used as negative control. At the
end of incubation, cells were washed 3 times in PBS containing
1% BSA and incubated for 2 h at room temperature with secondary
goat antimouse antibody Alexa 488 labeled (1:200) (Molecular
Probes, Invitrogen, Cergy Pontoise, France) for tubulin staining,
Alexa Fluor 546 phalloidin labeled (1:40) (Molecular Probes,
Invitrogen, Cergy Pontoise, France) for actin staining, and Hoechst
33342 labeled (1:5000) (Molecular Probes, Invitrogen, Cergy
Pontoise, France) for nuclear staining. At the end of incubation,
cells were washed once in PBS and mounted in fluorescent
mounting medium (Dako, Trappes, France). Images were acquired
using a Nikon TE2000E fluorescent microscope (Nikon, Champigny-
sur-Marne, France) equipped with a Nikon DXM1200F digital
camera.
Tubulin Binding Assay. Sheep brain tubulin was purified
according to the method of Shelanski et al.32 by two cycles of
assemblysdisassembly and then dissolved in the assembly buffer
containing 0.1 M MES, 0.5 mM MgCl2, 1 mM EGTA, and 1 mM
GTP, pH 6.6 (the concentration of tubulin was about 2-3 mg/
mL). Tubulin assembly was monitored and recorded continuously
by turbidimetry at 350 nm in a UV spectrophotometer equipped
with a thermostatted cell at 37 °C. The IC50 value of each compound
was determined as the concentration which decreased the maximum
assembly rate of tubulin by 50% compared to the rate in the absence
of compound. The IC50 values for all compounds were compared
to the IC50 of colchicine, measured the same day under the same
conditions.
Experimental Glioma Assay. U87 human glioma cells (Ameri-
can type Culture Collection) were maintained in DMEM with 10%
FBS, antibiotics, and L-glutamine. Fertilized chicken eggs (Earl
Morizeau, Dangers, France) were handled as described.33 On
embryonic day 10, 5 × 106 U87 cells in 20 µL of medium were
deposited on chick chorio-allantoic membrane (CAM). On day 2
of tumor development, size matched tumors were divided into
control and treatment groups. Twenty µL of each tested compound
(4f and colchicine at 10 µM and NAC at 100 µM) were deposited
locally on the tumor once a day (from day 3 to day 6 of tumor
growth). Controls received solvent of the corresponding drug.
Tumor size was calculated from the following formula: V ) 4/3πr3,
with r ) 1/2ꢀ(length × width). Quantification of drug effect on
tumor size was determined in six representative tumors per group.
Digital photos were taken under a stereomicroscope (Nikon
SMZ1500).
(R,S)-5,6,8-Trimethyl-5,8-dihydroindolo[2,3-d][2]benzazepin-
7(6H)-one (13). A solution of lactam 4b (13 mg, 0.05 mmol) in
dry THF (0.5 mL) was added at 0 °C under argon to a suspension
of sodium hydride (9 mg, 0.2 mmol, 60% in oil) in dry THF (0.5
mL). The reaction mixture was stirred for 30 min and methyl iodide
(20 µL, 0.3 mmol) was added. The reaction mixture was stirred at
rt for 2 h. Methanol and silica were added and the solvent and
excess methyl iodide were evaporated in vacuo. The residue was
purified by flash chromatography on silica gel (heptane/AcOEt 70/
1
30) to give 13 as a white solid (9 mg, 70% yield). H NMR (250
MHz, CDCl3) δ: 8.03 (m, 2H), 7.51 (m, 5H), 7.25 (m, 1H), 4.68
(q, J ) 7.3 Hz, 1H), 4.12 (s, 3H), 3.03 (s, 3H),1.81 (d, J ) 7.3 Hz,
3H). 13C NMR (75 MHz, CDCl3) δ: 162.4, 138.8, 138.1, 133.4,
131.4, 128.4, 127.8, 126.4, 124.7, 124.1, 123.3, 121.3, 120.9, 117.4,
110.3, 52.1, 32.0, 26.8, 15.0. FTIR (ν/cm-1) 1627. HRMS (ESI+)
calcd for [C19H18N2ONa]+ 313.1317, found 313.1280. Anal.
(C19H18N2O·0.4CH3CO2C2H5 ·0.4H2O) C, H, N.
(R,S)-5,8-Dimethyl-5,8-dihydroindolo[2,3-d][2]benzazepin-7(6H)-
one (14). Methyl iodide (31 µL, 0.5 mmol) was added to a solution
of lactam 4b (13 mg, 0.05 mmol) in toluene (0.5 mL) and water
(0.25 mL) containing a catalytic amount of TBAB. The reaction
mixture was stirred at rt for 2 h and extracted with DCM (3 × 5
mL). The combined organic layers were dried (MgSO4), filtered,
and evaporated in vacuo. The resulting residue was purified by flash
chromatography on silica gel (heptane/AcOEt 90/10 to 50/50) to
give compound 14 as a white solid (6.5 mg, 47% yield). 1H NMR
(250 MHz, CDCl3) δ: 8.06 (d, J ) 8.0 Hz, 1H), 8.00 (d, J ) 7.3
Hz, 1H), 7.49 (m, 5H), 7.25 (m, 1H), 6.14 (br s, 1H), 4.37 (qu, J
) 7.3 Hz, 1H), 4.15 (s, 3H), 1.79 (d, J ) 7.3 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ: 163.0, 140.1, 139.0, 133.4, 129.7, 128.9, 127.7,
126.8, 125.0, 124.3, 123.1, 121.6, 121.6, 118.8, 110.4, 48.0, 31.8,
17.2. FTIR (ν/cm-1) 3196, 1657. HRMS (ESI+) calcd for
[C18H17N2O]+ 277.1341, found 277.1338.
Cell Culture and Proliferation Assay. Cancer cell lines were
obtained from the American type Culture Collection (Rockville,
MD) and were cultured according to the supplier’s instructions.
Briefly, human KB epidermal carcinoma cells were grown in
Eagle’s minimal essential medium (EMEM) containing 4.5 g/L
glucose supplemented with 10% fetal calf serum (FCS) and 1%
glutamine. A549 lung carcinoma, MDA-MB231, MDA-MB435,
and MCF7 breast carcinomas and mouse B16F10 melanoma cells
were grown in Dulbecco minimal essential medium (DMEM)
containing 4.5 g/L glucose supplemented with 10% FCS and 1%
glutamine. Human K562 leukemia and HCT116 colorectal carci-
noma cells were grown in RPMI 1640 containing 10% FCS and
1% glutamine. All cell lines were maintained at 37 °C in a
humidified atmosphere containing 5% CO2. Cell viability was
assessed using Promega CellTiter-Blue TM reagent according to
the manufacturer’s instructions. Briefly, cells were seeded in 96-
well plates (5 × 103 cells/well) containing 50 µL growth medium.
After 24 h of culture, the cells were supplemented with 50 µL of
the tested compound dissolved in DMSO (less than 0.1% in each
preparation). After 72 h of incubation, 20 µL of resazurin was added
for 2 h before recording fluorescence (λex ) 560 nm, λem ) 590
nm) using a Victor microtiter plate fluorimeter (Perkin-Elmer,
USA). The IC50 corresponds to the concentration of the tested
compound that caused a decrease of 50% in fluorescence of drug-
treated cells compared with untreated cells. Experiments were
performed in triplicate.
Acknowledgment. We thank N. Hajem for technical as-
sistance and the Institut de Chimie des Substances Naturelles
for funding and fellowships (L.K., S.B., J.-M.L).
Cell Cycle Analysis. Exponentially growing cancer cells (K562,
MCF7, MDA-MB231) were incubated with tested compound or
DMSO for 6, 16, and/or 24 h. Cell-cycle profiles were determined

C5-Alkylated Indolobenzazepinones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3421
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Supporting Information Available: Physical and spectroscopic
data for new compounds. Cytometric flow results with compound
4f. Elemental analysis data. This material is available free of charge
via the Internet at http://pubs.acs.org.
(17) Peukert, S.; Brendel, J.; Hemmerle, H.; Kleemann, H.-W. PCT Int.
Appl. WO 0248131 A1 20020620, 2002.
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