Indolobenzazepin-7-ones and 6-, 8-, and 9-membered ring derivatives as tubulin polymerization inhibitors: Synthesis and structure-activity relationship studies

Article abstract of DOI:10.1021/jm900476c

Several small weight indole derivatives (D-64131, D-24851, BPR0L075, BLF 61-3, and ATI derivatives) are potent tubulin polymerization inhibitors and show nanomolar antiproliferative activity. Among them, indolobenzazepin-7-ones were recently disclosed as

Full text of DOI:10.1021/jm900476c

5916 J. Med. Chem. 2009, 52, 5916–5925
DOI: 10.1021/jm900476c
Indolobenzazepin-7-ones and 6-, 8-, and 9-Membered Ring Derivatives as Tubulin Polymerization
Inhibitors: Synthesis and Structure-Activity Relationship Studies
†
†
‡
Aurelien Putey, Florence Popowycz, Quoc-Tuan Do, Philippe Bernard,*,‡ Sandeep K. Talapatra,§ Frank Kozielski,*,§
ꢀ
Carlos M. Galmarini,*, and Benoıt Joseph*,†
†Institut de Chimie et Biochimie Moleꢀculaires et Supramoleꢀculaires, UMR-CNRS 5246, Laboratoire de Chimie Organique 1, Universiteꢀde Lyon,
Universiteꢀ Claude Bernard-Lyon 1, Ba^timent Curien, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France, ‡GreenPharma SA,
3 Alleꢀe du Titane, 45100 Orleꢀans, France, §The Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow G61 1BD, U.K., and
ENS-CNRS UMR 5239, UFR de Meꢀdecine Lyon-Sud, 165 chemin du Grand Revoyet, BP 12, 69921 Oullins, France
Received April 14, 2009
Several small weight indole derivatives (D-64131, D-24851, BPR0L075, BLF 61-3, and ATI deri-
vatives) are potent tubulin polymerization inhibitors and show nanomolar antiproliferative activity.
Among them, indolobenzazepin-7-ones were recently disclosed as potent antimitotic agents. In an effort
to improve this structure, we prepared new derivatives in order to evaluate their antiproliferative
activity. 5,6,7,9-Tetrahydro-8H-indolo[2,3-e][3]benzazocin-8-one (1m) was found to be the most potent
derivative inhibiting the cell growth of several cancer cell lines in the lower nanomolar range.
Introduction
site.9 Among them, D-64131 (IC50 U373 = 72 nM, IC50
inhibition of tubulin polymerization (ITPa) = 0.53 μM),10
D-24851 (IC50 HT29=72 nM, IC50 MDA-MB231=74 nM,
andIC50 ITP = 1.0 μM),11 BPR0L075(IC50 KB = 4 nM, IC50
H460 = 7 nM, and IC50 ITP = 2.8 μM),12 BLF 61-3 (IC50
MCF7 = 45 nM and IC50 ITP = 1.6 μM),13 and 3-arylthioin-
dole (ATI) derivative (IC50 MCF7 = 13 nM and IC50 ITP=
2.0 μM)14 displayed nanomolar cell growth inhibition
(Figure 1). The indole scaffold has also been investigated
intensively due to the advantages of easier access and lower cost.
These initial results highlighted the indole nucleus as a valuable
pharmacophore for potent tubulin polymerization inhibitors.
Previously, we reported the synthesis of indolobenzazepin-
7-ones 1a-1f and derivatives 2a-3a via an original C-3 indole
direct arylation mediated by palladium (Figure 1).15 An initial
screening showed antiproliferative properties for this scaf-
fold.15b Similarly, R. Dodd et al. have prepared the same
heterocyclic core by a Suzuki coupling reaction between ethyl
3-iodoindole-6-carboxylates and appropriate R-alkylbenzyla-
mino o-boronic acids followed by final lactamization. These
compounds displayed potent inhibition of tubulin polymeri-
zation and high antiproliferative activities in vitro and in
cell-based assays (i.e., (S)-5-ethyl indolobenzazepin-7-one:
IC50 MCF7 = 40 nM, IC50 HCT116 = 42 nM, and IC50
ITP = 1.9 μM) (Figure 1).16 These data prompted us to
evaluate indolobenzazepinones 1a-1f and derivatives
2a-3a, and we initiated a structure-activity relationship
study to provide more derivatives in order to compare their
antiproliferative and tubulin inhibitory activities with those
obtained for (S)-5-ethyl indolobenzazepinone and 1a-1f.
Present in all eukaryotic cells, microtubules are primordial
components of cell structure, which play a pivotal role in a
wide number of essential cellular functions, such as morpho-
genesis, motility, cell division, and intracellular transport.1
Microtubules are composed of R- and β-tubulin heterodimers
characterized by a highly dynamic behavior (fluctuations
between phases of elongation and shortening).2 Drugs inter-
fering with microtubules/tubulin dynamics or other proteins
of the mitotic spindle (kinases, kinesins) are called antimitotic
agents.3 The tubulin-targeting agents are important in cancer
therapy and have been grouped into two main classes:4 (1)
tubulin stabilizing derivatives such as paclitaxel, docetaxel,
and epothilone B,5 and (2) tubulin polymerization inhibitors
with different mechanisms of action6,7 (t-BCEU, T138067,
macrocyclic polyethers, depsipeptides, maytansine, rhizozine,
colchicine, and combretastatin A48) (Figure 1).
In the past decade, an increasing number of small molecular
weight indole derivatives have been described as potent tubu-
lin polymerization inhibitors through binding to the colchicine
*To whom correspondence should be addressed. P.B. (chemo-
informatic): phone, þ33 (0)2 38 25 99 80; fax, þ33 (0)2 38 25 99 65;
e-mail, philippe.bernard@greenpharma.com. F.K. (biology, tubuline,
colchicine): phone, þ44 (0)141 330 3186; fax, þ44 (0)141 942 6521;
e-mail, f.kozielski@beatson.gla.ac.uk. C.M.G. (biology, MTT, cell
cycle): phone, þ33 (0)2 98 29 23 39; fax, þ33 (0)2 98 29 25 26; e-mail,
cmgalma@hotmail.com; B.J. (chemistry): phone, þ33 (0)4 72 44 81 35;
fax, þ33 (0)4 72 43 12 14; e-mail, benoit.joseph@univ-lyon1.fr.
a Abbreviations: U373, human glioblastoma-astrocytoma cell line;
ITP, inhibition of tubulin polymerization; HT29, human colon cancer
cell line; MDA-MDB231, human breast carcinoma cell line; KB,
vincristine-resistant human cancer cell line; H460, human lung cancer
cell line; ATI, 3-arylthioindole; MCF7, human breast cancer cell line;
DMAP, 4-(dimethylamino)pyridine; EDCI, 1-(3-dimethylaminopro-
pyl)-3-ethylcarbodiimide hydrochloride; DMF, N,N-dimethylforma-
mide; 5-FU, 5-fluorouracil; PDB, protein data bank; PE, petroleum
ether; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide.
Results and Discussion
Chemistry. The syntheses of 6-alkyl- or 6-alkenylindolo-
benzazepin-7-ones 1g-1k are depicted in Scheme 1. The
starting material 5 was prepared from 1-(ethoxymethyl)-
1H-indole-2-carboxylic acid 4 as previously described.15
r
pubs.acs.org/jmc
Published on Web 09/10/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5917
Figure 1. Structures of various known antimitotic agents and indolobenzazepin-7-ones 1a-1f and derivatives 2a and 3a.
Scheme 1a
a Reagents and conditions: (i) NaH, R1I or R1Br, THF, rt, 4-60 h, 6g (EtI, 20 h) = 87%, 6h (PrI, 60 h) = 90%, 6i (i-PrI, 80 °C, sealed tube, 20 h) =
77%, 6j (BrCH2-CHdC(Me)2, 4 h) = 91%, 6k (ClCH2-CH2-N(Me)2, 4 h) = 74%; (ii) 1 N HCl, dioxane, 80 °C, 2 h, 1g (Et) = 93%, 1h (Pr) = 95%,
1i (i-Pr) = 57%, 1j (CH2-CHdC(Me)2) = 83%, 1k (CH2-CH2-N(Me)2) = 44%.
N-Alkylation of 5 was performed in the presence of sodium
hydride and halogen halides (iodoethane, iodopropane, 2-
iodopropane, 1-bromo-3-methylbut-2-ene, and 1-chloro-
2-(dimethylamino)ethane) to afford derivatives 6g-6k in fair
yields. Final deprotectionof6 in acidic medium gave 1g-1k in
83-95% yield. Lower or higher membered ring analogues
1l-1o were then prepared in four steps from 4 through the key
intramolecular direct arylation reaction (Scheme 2).17 Acid 4
was first treated with various amines 7l-7n,18 DMAP, and
EDCI to give amides 8l-8n. Boc protection (Boc2O, DMAP)
of 8 was carried out to afford 9l-9n in near-quantitative
yields. N-Methylation of 8m was also performed to obtain
compound 9o in 99% yield. The cyclization mediated by Pd(0)
of 9l was performed in the presence of 20 mol % of Pd(OAc)2,
PPh3 (0.4 equivalent), and Ag2CO3 (2 equivalents) in DMF at
100 °C for 1 h to give indoloquinolin-6-one 10l in 71% yield.
Lower amounts of Pd(OAc)2 led to a decrease of product
yield. For 9m-9o, the direct arylation on C-3 position of
the indole occurred in the presence of 5 mol % of Pd(OAc)2 at
140 °C for 1-3.5 h to give 10m-10o in 47-97% yield. In all
cases except for 9o, the Boc protecting group on amide is
required to get the cyclized compound. HCl hydrolysis of 10
eventually led to final compounds 1l-1o. Low deprotection
yields were observed with 8-membered ring compounds 1m
and 1o because of the opening of the lactam ring under acidic
conditions. Finally, the same synthetic strategy was applied to
obtaincompounds 1p-1s withthe methoxysubstituent on the
benzene moiety (Scheme 3). Amides 12p-12s were prepared
from 4 and 2-iodobenzylamines18b,19 11p-11s, then Boc
derivatives 13p-13s were obtained quantitatively. Intra-
molecular arylation of 13 was effective in the presence of
20 mol % of Pd(OAc)2 to give 14 in 40-83% yield. Cycliza-
tion assays on 13 with lower quantities of Pd(OAc)2 led to
low yields. Removal of both protecting groups on 14 was
achieved in acidic conditions (1 N HCl, 1,4-dioxane) to
afford 1p-1s.

5918 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Putey et al.
Biological Evaluation: In Vitro Cell Growth Inhibition. All
of the reported compounds were first evaluated for cytotoxi-
city in the human breast cancer cell line MCF7. Paclitaxel,
vinorelbine, and 5-fluorouracil (5-FU) were included in the
assays as examples of antitumor agents currently used in the
clinic. IC50 values for cell growth inhibition are shown in
Tables 1 and 2. The core indolobenzazepin-7-one compound
1a displayed submicromolar activity (IC50=0.3 μM) as
already observed by Dodd et al.16 When a methoxy group
was introduced at the C-4 position of the indole group (1b),
the resulting compound presented a cell growth inhibition
activity very similar to that of 1a (IC50=0.4 μM). The pre-
sence of the same group at the C-5 or C-6 position of the
indole group (1c and 1d) led to decreasing antiproliferative
activity (IC50 = 3.1-3.2 μM). Methylation of the amide
nitrogen atom leads to compounds as potent as 1a (1e
IC50 =0.3 μM), and a slight loss of activity was observed
for 1f. Homologation of the alkyl chain in this position
(1g-1i) resulted in a drastic reduction of cytotoxic activity
(IC50>30 μM). However, the presence of 3-methylbut-2-enyl
chain or N,N-dimethylaminoethyl chain at the same position
(1j and 1k) led to modest antiproliferative activity (IC50
=
6.9-18.6 μM). Seven-membered ring compound 1a is
approximately 22 times more active than the quinolin-6-
one derivative 1l (IC50=6.5 μM). Eight-membered ring
compound 1m produced similar cytotoxic activity in
MCF7 cells (IC50 = 0.2 μM) compared to that of 1a and
1e (IC50 = 0.3 μM). The nine-membered heterocyclic deri-
vative 1n showed micromolar cell growth inhibition and was
the least active compound of all homologues tested.
N-Methylation of 1m induced a loss of antiproliferative
activity in MCF7 cells (1o, IC50 = 2 μM). Methoxy groups
on the benzene cycle led to decreasing antiproliferative
activity. Compounds 1p-1r were more than 100-fold less
active than 1a. It should be noted that the presence of three
methoxy groups in 1s induced a higher cell growth inhibitory
activity with IC50 values at the low micromolar range and
only 17-fold higher than those obtained with 1a. The indole
moiety is crucial for the retention of the antiproliferative
activity. Benzo[b]thieno[2,3-d][2]benzazepin-7-one 2a or
pyrrolo[2,3-d][2]benzazepin-7-one 3a were considerably less
active (IC50 > 30 μM). As already mentioned by Dodd et al.,
N-alkylation of the indole nitrogen had a detrimental effect
on activity (IC50 > 30 μM).16
Scheme 2a
The most potent agents (1a, 1b, 1e, and 1m), 1c, 1d, and 1o
were next tested for their antiproliferative activity on a panel
of eight human cancer cell lines: sarcoma Mes-sa cells, breast
cancer T47D and UACC812 cells, lymphoma CEM cells,
lymphoma RL cells and colorectal cancer HCT116, SW48
and SW480 cells. As shown in Table 3, all seven compounds
exhibited similar cell growth inhibitory activities against all
cell lines as in MCF7 cells. Again, compound 1m showed the
highest IC50 values for all cancer cell lines tested except for
sarcoma Mes-sa cells.
a Reagents and conditions: (i) 2 M AlMe3 in toluene, CH2Cl2, -20 °C
to rt, 19 h, 8l = 98%; (ii) DMAP, EDCI, CH2Cl2, 0 °C to rt, 24 h, 8m =
98%, 8n = 97%; (iii) Boc2O, DMAP, MeCN, rt, 15 h, 9l-9n = 99%;
(iv) NaH, MeI, THF, rt, 4 h, 9o = 99%; (v) Pd(OAc)2 (20 mol %), PPh3
(0.4 equiv), Ag2CO3 (2 equiv), DMF, 100 °C, 1 h, 10l = 71%; (vi)
Pd(OAc)2 (5 mol %), PPh3 (0.1 equiv), Ag2CO3 (2 equiv), DMF, 140 °C,
10m = 97% (1 h), 10n = 47% (1 h), 10o = 87% (3.5 h); (vii) 1 N HCl,
dioxane, 80 °C, 1l = 70% (3 h), 1m = 28% (5 h), 1n = 67% (2 h), 1o =
51% (1 h).
Effect on Cell Cycle Progression. Compounds 1a, 1b, 1e,
and 1m were selected for cell cycle studies in MCF7, Mes-sa,
Scheme 3a
a Reagents and conditions: (i) DMAP, EDCI, CH2Cl2, 0 °C to rt, 24 h, 12p = 85%, 12q = 97%, 12r = 85%, 12s = 95%; (ii) Boc2O, DMAP, MeCN,
rt, 15 h, 13p-13s = 99%; (iii) Pd(OAc)2 (20 mol %), PPh3 (0.4 equiv), Ag2CO3 (2 equiv), DMF, 100 °C, 14p = 73% (1 h), 14q = 53% (23 h), 14r = 83%
(1 h), 14s = 69% (2 h); (iv) 1 N HCl, dioxane, 80 °C, 1p = 20% (3 h), 1q = 91% (3 h), 1r = 92% (1 h), 1s = 73% (1 h).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5919
Table 1. Cytotoxic Effects of Compounds 1a-1o, 2a, 3a, and 5 on MCF7 Cancer Cell Lines and Inhibition of Tubulin Polymerization (ITP)
compd
5-FU
vinorelbine
R1
R2
R3
R4
n
MCF7a IC50 ( SD (μM)b
ITPc ( SD (μM)d
0.3 ( 0.1
0.07 ( 0.01
0.003 ( 0.002
0.3 ( 0.1
0.4 ( 0.2
3.1 ( 1.2
3.2 ( 0.6
0.3 ( 0.1
3.1 ( 1.1
>30
paclitaxel
1a
1b
1c
1d
1e
1f
H
H
H
H
1
1
1
1
1
1
1
1
1
1
1
0
2
3
2
5.3 ( 1.3
6.1 ( 0.4
nie
OMe
H
H
H
H
OMe
H
H
H
H
OMe
H
H
6.2 ( 1.2
4.4 ( 0.9
3.7 ( 1.6
ndf
H
H
Me
Me
Et
i-Pr
Pr
OMe
H
H
H
1g
1h
1i
H
H
H
H
H
>30
>30
ni
nd
H
H
H
1j
H
H
H
-CH2-CHdC(Me)2
-CH2-CH2-N(Me)2
6.9 ( 2
9.1 ( 2.3
ni
1k
1l
H
H
H
18.6 ( 2.3
6.5 ( 0.7
0.2 ( 0.1
8 ( 1.4
2 ( 0.07
>30
>30
H
H
H
H
ni
1m
1n
1o
2a
3a
5
H
H
H
H
7.7 ( 1.2
13.0 ( 4.4
5.4 ( 1.3
14.9 ( 1.3
ni
H
H
H
H
Me
H
H
H
>30
ni
a MCF7 = breast cancer. b IC50 is the concentration of the test compound inducing 50% cell growth inhibition after 72 h of incubation. c ITP =
inhibition of tubulin polymerization. d IC50 is the concentration of compound required to inhibit 50% of the rate of microtubule assembly. e ni = no
inhibition. f nd = not determined.
Table 2. Cytotoxic Effects of Compounds 1p-1s on MCF7 Cancer Cell
Lines and Inhibition of Tubulin Polymerization
paclitaxel or vinorelbine treatment (data not shown). In
these cancer cell lines, compounds 1a, 1b, 1e, and 1m induced
similar levels of G2/M arrest. In order to detect whether the
G2/M arrest was specific to G2 or the mitotic phase, the
morphological features of compound-treated cells were then
analyzed by Hoechst33258/PI staining of cellular DNA. The
nuclei of untreated MCF7, Mes-sa, and HCT116 cells
showed the typical diffuse pattern of chromatin distribution
(data not shown), whereas cells treated with compound
derivatives became arrested in prometaphase as an effect of
perturbation of tubulin function by these compounds
(Figure 2).
MCF7a
ITPc ( SD
(μM)d
nie
In Vitro Tubulin Polymerization. We investigated the in-
hibition of tubulin polymerization of all compounds (except
for 1g and 1i) using in vitro turbidimetric measurements
of microtubule assembly (Tables 1 and 2). Out of 20 com-
pounds tested, 9 did not significantly influence tubulin
polymerization. The other 11 compounds inhibited assembly
into microtubules with estimates of IC50 values in the range
from 3.7 μM to 15.9 μM. The rate as well as the final extent
of assembly were reduced by most of these compounds. In
agreement with other indole-based inhibitors, inhibition of
different tumor cell lines in the nanomolar range corre-
sponds to IC50 values in the lower micromolar range in in
vitro assays.5-10 The in vitro data are in good agreement with
the data obtained from cell-based assays: compounds that do
not inhibit the MCF7 tumor cell line (1h, 1k, 2a, 3a, 5, 1p, 1q,
compd
R1
R2
R3
IC50 ( SD (μM)b
1p
1q
1r
1s
OMe
H
H
H
>30
>30
>30
5 ( 4
H
OMe
OMe
OMe
ni
H
OMe
OMe
OMe
ni
9 ( 0.6
a MCF7 = breast cancer. b IC50 is the concentration of the test
compound inducing 50% cell growth inhibition after 72 h of incubation.
c ITP = inhibition of tubulin polymerization. d IC50 is the concentration
of compound required to inhibit 50% of the rate of microtubule
assembly. e ni = no inhibition.
and HCT116 tumor cell lines. Cell cycle analysis (Table 4)
following treatment with 10 μM of each compound showed
that most of the cells were arrested in the G2/M phase after
24 h of treatment. Similar features were observed after

5920 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Putey et al.
Table 3. Antiproliferative Activities of 1a-e, 1m, and 1o against Various Cancer Cell Lines
IC50 ( SD (μM)a,b
compd
Mes-sa
T47D
UACC812
RL
CEM
HCT116
2.6 ( 1
0.001 ( 0.001
0.4 ( 0.09
0.5 ( 0.09
1.9 ( 0.1
SW48
4.4 ( 1
0.001 ( 0.001
0.2 ( 0.06
0.5 ( 0.04
2.1 ( 0.4
SW480
5-FU
paclitaxel
1.1 ( 0.4
<0.001
0.6 ( 0.1
0.004 ( 0.002
0.6 ( 0.1
0.6 ( 0.1
ndc
3.1 ( 1
0.7 ( 0.1
0.76 ( 0.19
2.7 ( 1
nd
3.2 ( 1
125 ( 35
2.1 ( 1
0.003 ( 0.001
0.22 ( 0.005
0.5 ( 0.007
nd
0.002 ( 0.001
0.2 ( 0.06
0.2 ( 0.03
1.4 ( 0.1
0.002 ( 0.001
0.4 ( 0.1
0.7 ( 0.1
3.1 ( 1.2
5.4 ( 3.3
0.3 ( 0.2
0.18 ( 0.02
19 ( 1.4
1a
1b
1c
1d
1e
1m
1o
0.3 ( 0.08
0.5 ( 0.1
5 ( 0.7
3.5 ( 0.6
0.4 ( 0.2
21.5 ( 4.9
1.4 ( 0.3
nd
nd
nd
2.6 ( 0.8
110 ( 21
2.3 ( 0.3
0.6 ( 0.3
0.1 ( 0.005
nd
0.81 ( 0.3
0.26 ( 0.05
nd
0.21 ( 0.007
0.07 ( 0.03
nd
0.1 ( 0.02
0.05 ( 0.03
1.5 ( 0.5
0.2 ( 0.09
0.08 ( 0.03
1 ( 0.03
0.2 ( 0.05
0.05 ( 0.03
0.9 ( 0.2
a IC50 is the concentration of the test compound including 50% cell growth inhibition after 72 h of incubation; Mes-sa = uterine sarcoma; T47D,
UACC812 = breast cancer; RL = lymphoma; CEM = T-cell acute lymphoblastic leukemia; HCT116, SW48, SW480 = human colon cancer. b Values
are the means of three different experiments ( SD. c nd = not determined.
Table 4. Percentage of MCF7, Mes-sa and HCT116 Cancer Cell Lines in the Different Phases after 24 h of Treatment with Compounds 1a, 1b, 1e,
and 1m
MCF7
S
Mes-sa
S
HCT116
S
compda
G1
G2/M
G1
G2/M
G1
G2/M
control
1a
1b
47.6 ( 7
37.9 ( 6
4.2 ( 4.7
9 ( 0.3
5.3 ( 1
16 ( 4.4
14.4 ( 3.2
66.6 ( 4.8
66 ( 0.9
67.1 ( 1.8
81 ( 6.1
61 ( 5
7 ( 2
9 ( 3
9 ( 1
9 ( 2
30 ( 4
5 ( 1
10 ( 5
2 ( 1
10 ( 2
87 ( 4
82 ( 2
89 ( 2
72 ( 9
62 ( 6
5 ( 1
5 ( 0.4
2 ( 1
25 ( 8
12 ( 3
94 ( 1
92 ( 1
96 ( 2
95.3 ( 4
29.6 ( 8.8 (
27.5 ( 2.8
27.5 ( 2.8
2.3 ( 1.7
2 ( 0.2
3 ( 0.4
2 ( 2
1e
1m
18 ( 7
3.3 ( 0.5
13.7 ( 0.4
a Compounds were tested at 10 μM.
Figure 2. Prometaphase arrest induced by treatment of HCT116
cells with 1a at 10 μM.
1r, and 1s) do not inhibit or only weakly inhibit tubulin
polymerization. Compounds that inhibit MCF7 in the na-
nomolar range (1a, 1b, 1e, and 1m) all inhibit tubulin
polymerization in the low μmolar range indicating that the
in vitro assay can predict whether or not a compound will be
active in cell-based assays.
Colchicine-Binding Site. To investigate whether our com-
pounds bind in or close to the colchicine-binding domain, we
performed competition assays in the presence of fluorescent
colchicine using compounds 1e, 1m, and 1p. Compound 1e is
one of the potent compounds in inhibiting tubulin polymer-
ization, whereas 1m is the most potent compound in cell-
based assays. Compound 1p has the same scaffold as the
other two compounds but did not inhibit tubulin polymer-
ization. The results of the competition assays are presented in
Figure 3. As expected, vinblastine, which binds to the vinca
domain does not compete with colchicine, indicated by the
same fluorescence intensity as that for fluorescent colchicine
alone. Colchicine competes with its fluorescent analogue
Figure 3. Competition assays of different tubulin-binding drugs
with fluorescent colchicine. The fluorescence intensity at 516 nm is
plotted against the elution volume. Fluorescent colchicine in the
absence of any inhibitor (red), colchicine (positive control, dark
blue), vinblastine (negative control, green), and compounds 1e
(black), 1m (yellow), and 1p (light blue).
indicated by the disappearance of the intensity peak. Com-
pound 1p, which does not inhibit tubulin polymerization
does not compete with fluorescent colchicine. Compounds
1e and 1m do compete with fluorescent colchicine as indi-
cated by the loss of fluorescence intensity, and interestingly,
the extent of competition of the two compounds correlates
with their respective IC50 values obtained from inhibition of
tubulin polymerization assays. We can conclude that our

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5921
Figure 4. (A) Proposed binding mode for 1a (cyan), lm (green), and 1n (magenta). (B) Binding mode of cocrystallized colchicine (green). The
green dotted ellipse highlights the zone of the binding site where substitutions are possible, while the red one locates the steric hindrance part of
the cavity. Pink dotted lines represent possible hydrogen bond interactions between the protein and the different ligands. Residue numbers are
those used by Ravelli et al.7
active compounds bind in or close to the colchicine binding
domain.
5,6,7,9-tetrahydro-8H-indolo[2,3-e][3]benzazocin-8-one 1m is
the mostpotent antiproliferative agent across a widevariety of
cancer cell lines. As mentioned by Dodd et al., these com-
pounds are antimitotic agents acting through the colchicine
binding site of the tubulin. The docking studies suggest that
the indole part of 1m is likely overlaid with the aryl moiety of
the colchicine, and the carbonyl group of the amide may form
the single H-bond with the nitrogen of the Asn-101 side chain
of the colchicine binding site. Structural modifications of the
basic 5,6,7,9-tetrahydro-8H-indolo[2,3-e][3]benzazocin-8-one
core are under investigation to improve the inhibitory po-
tency, and will be reported in due course.
Docking and Molecular Modeling. To validate this hypoth-
esis, compounds 1a, 1m, and 1n were docked into the
colchicine binding site of tubulin. The tubulin crystal struc-
ture was retrieved from the Protein Data Bank (PDB). The
accession code 1SA0 corresponds to the R,β-tubulin hetero-
dimer with bound colchicine.7 Docking studies were under-
taken with FlexX v2.2 with default settings in order to
predict the binding mode of our ligands. It was found that
the indole part of 1m is probably overlaid with the trimethox-
yphenyl group of the colchicine derivative cocrystallized
with tubulin as shown in Figure 4. The carboxyl group of
the amide may form an H-bond with the nitrogen of the
Asn-101 side chain. There is no interaction of the nitrogen
amide of indolobenzazepinone derivatives with the protein.
Alkylation at that position decreased the activity of most
compounds in the MCF7 assays, while its influence on
tubulin polymerization is not obvious (Table 1). Methoxy
groups on R1, R2, and R3 did not give benefits in terms of
biological activities. From the results in Table 2, the added
methoxy group of the other phenyl group is damaging to the
compound’s activities even if the trimethoxy derivative 1s
displayed micromolar antiproliferative property.
Experimental Section
Chemistry. General Methods. Commercial reagents (Fluka,
Aldrich) were used without purification. Solvents were distil-
lated prior to use. The light petroleum ether (PE) refers to the
fraction boiling at 40-60 °C. Melting points were determined
using a Buchi capillary instrument and are uncorrected. IR
spectra were recorded on Perkin-Elmer Spectrum One. NMR
spectra were recorded at 300 K on a Bruker Avance spectro-
meter at 300 MHz for 1H and 75 MHz for 13C. Chemical shifts
are reported in ppm (δ) relative to tetramethylsilane as an
internal standard. Mass spectra were recorded with a Perkin-
Elmer SCIEX API spectrometer. Elemental analyses were per-
formed on a Thermoquest Flash 1112 series EA analyzer.
Elemental analyses were found to be within ( 0.4% of the
theoretical values. Purity of tested compounds was >95%. Thin
layer chromatography (TLC) analyses were conducted on alu-
minum sheet silica gel Merck 60F254. The spots were visualized
using ultraviolet light. Flash chromatography was carried out
on silica gel 60 (40-63 μm, Merck) using the indicated solvents.
Compounds 1a-1f, 2a, 3a, 4 and 5 were prepared as previously
described.15
According to molecular modeling predictions, there may
be two possible H-bond donors from the protein: the NH of
the Asn-101 side chain (Figure 4A) and the NH of the Val-
181 main chain (Figure 4B). This observation raises the
possibility of an additionnal H-bond formation with the
protein (NH Val-181) and the 5,6,7,9-tetrahydro-8H-indolo-
[2,3-e][3]benzazocin-8-one scaffold.
Conclusions
1-(Ethoxymethyl)-N-(2-iodophenyl)-1H-indole-2-carboxamide
(8l). At -20 °C and under inert atmosphere, a 2.0 M AlMe3
solution in toluene (5.05 mL, 10.11 mmol) diluted in CH2Cl2
(16 mL) was added dropwise to a solution of 2-iodoaniline 7l
(1.77 g, 8.08 mmol) in CH2Cl2 (5 mL). The solution was stirred
at -20 °C for 45 min then abandoned to reach 0 °C. A solution
of ethyl 1-(ethoxymethyl)-1H-indole-2-carboxylate 5 (500 mg,
2.02 mmol) in CH2Cl2 (4 mL) was added dropwise. The final
solution was stirred at room temperature for 19 h. The medium
Synthesis and structure-activity relationship studies for
indolobenzazepinone as anticancer agents were carried out.
The SAR information indicated that the introduction of the
methoxy group on benzene or indole moieties, or alkylation of
the amide nitrogen did not improve the antiproliferative
activity compared with that of the unsubstituted compound.
Compared to six-, seven- and nine-membered ring derivatives,

5922 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Putey et al.
was carefully acidified at 0 °C by the addition of 1 N HCl (45 mL)
then stirred for 10 min. The medium was diluted by the addition
of H2O (10 mL) and extracted by CH2Cl2 (3 ꢀ 50 mL). The
organic phases were combined, dried over Na2SO4, filtered, and
evaporated in vacuo. The crude solid was recrystallized from
MeOH to give 8l (836 mg, 98%). Mp 147-148 °C (MeOH). IR
(KBr): ν 3278, 3026, 2978, 1648, 1522, 1433, 1312, 1292, 1086,
749 cm-1. 1H NMR (CDCl3): δ 1.15 (t, 3H, J = 7.0 Hz, CH3),
3.58 (q, 2H, J = 7.0 Hz, CH2), 6.05 (s, 2H, CH2), 6.91 (dt, 1H,
J = 1.3, 8.0 Hz, Harom), 7.21-7.26 (m, 2H, Harom), 7.37-7.44
(m, 2H, Harom), 7.62 (d, 1H, J = 8.5 Hz, Harom), 7.72 (d, 1H, J =
7.9 Hz, Harom), 7.84 (dd, 1H, J = 1.3, 8.0 Hz, Harom), 8.37 (dd,
1H, J = 1.3, 8.3 Hz, Harom), 8.41 (broad s, 1H, NH). 13C NMR
(CDCl3): δ 15.2 (CH3), 64.2 (CH2), 73.7 (CH2), 90.5 (C), 107.4
(CH), 111.5 (CH), 121.7 (CH), 122.1 (CH), 122.3 (CH), 125.4
(CH), 126.3 (CH), 126.5 (C), 129.5 (CH), 131.8 (C), 138.4 (C),
139.1 (CH), 139.7 (C), 160.2 (CO). MS (IS): m/z 421 (M þ Hþ).
Anal. (C18H17IN2O2) C, H, N.
by flash chromatography (eluent: PE/EtOAc 95:5 to 9:1) to give 9l
(980 mg, 99%) as an oil. IR (film): ν 3054, 2983, 1737, 1678, 1469,
1265, 1151, 1096, 741 cm-1. 1H NMR (CDCl3): δ 1.15 (t, 3H, J =
7.2 Hz, CH3), 1.31 (s, 9H, 3 CH3), 3.50-3.59 (m, 2H, CH2),
5.76-5.87 (m, 2H, CH2), 7.08 (dt, 1H, J = 1.3, 7.9 Hz, Harom),
7.17-7.23 (m, 2H, Harom), 7.33-7.48 (m, 3H, Harom), 7.59 (d, 1H,
J = 8.5 Hz, Harom), 7.66 (d, 1H, J = 7.9 Hz, Harom), 7.94 (dd, 1H,
J = 1.3, 7.9 Hz, Harom). 13C NMR (CDCl3): δ 15.1 (CH3), 27.8
(3CH3), 64.0 (CH2), 73.6 (CH2), 83.9 (C), 99.6 (C), 110.6 (CH),
111.3 (CH), 121.4 (CH), 122.5 (CH), 125.4 (CH), 126.3 (C), 129.3
(CH), 129.8 (CH), 129.9 (CH), 132.5 (C), 139.2 (C), 140.0 (CH),
141.8 (C), 152.3 (CO), 164.2 (CO). MS (IS): m/z 521 (M þ Hþ).
Anal. (C23H25IN2O4) C, H, N.
tert-Butyl-{[1-(ethoxymethyl)-1H-indol-2-yl]carbonyl}[2-(2-
iodophenyl)ethyl]carbamate (9m). According to the procedure
described for the synthesis of 9l, compound 9m was prepared
from 8m in 99% yield as an oil (chromatography eluent: PE/
EtOAc 9:1). IR (film): ν 3056, 2977, 1731, 1667, 1522, 1368,
1318, 1144, 743 cm-1. 1H NMR (CDCl3): δ 1.15 (t, 3H, J = 7.2
Hz, CH3), 1.16 (s, 9H, 3CH3), 3.20 (t, 2H, J = 7.1 Hz, CH2), 3.54
(q, 2H, J = 7.2 Hz, CH2), 4.05 (t, 2H, J = 7.1 Hz, CH2), 5.77 (s,
2H, CH2), 6.73 (d, 1H, J = 0.5 Hz, Harom), 6.91 (td, 1H, J = 1.9,
7.2 Hz, Harom), 7.17 (t, 1H, J = 7.0 Hz, Harom), 7.27-7.36 (m,
3H, Harom), 7.57 (d, 1H, J = 8.3 Hz, Harom), 7.61 (d, 1H, J = 7.9
Hz, Harom), 7.83 (dd, 1H, J = 1.1, 7.9 Hz, Harom). 13C NMR
(CDCl3): δ 15.0 (CH3), 27.5 (3 CH3), 39.9 (CH2), 45.5 (CH2),
63.9 (CH2), 73.5 (CH2), 83.0 (C), 100.7 (C), 108.1 (CH), 111.0
(CH), 121.2 (CH), 122.0 (CH), 124.8 (CH), 126.3 (C), 128.3
(CH), 128.4 (CH), 130.5 (CH), 133.6 (C), 138.7 (C), 139.5 (CH),
141.4 (C), 153.5 (C), 165.8 (CO). MS (IS): m/z 549 (M þ Hþ).
Anal. (C25H29IN2O4) C, H, N.
1-(Ethoxymethyl)-N-[2-(2-iodophenyl)ethyl]-1H-indole-2-car-
boxamide (8m). Under argon atmosphere, indole 4 (940 mg,
4.29 mmol), DMAP (0.52 g, 4.29 mmol), and EDCI (0.91 g,
4.72 mmol) were added to a solution of amine 7m (4.72 mmol) in
CH2Cl2 (45 mL) at 0 °C. The mixture was stirred for 4 h at 0 °C
then for 20 h at room temperature. The mixture was acidified by
the addition of a 6 N HCl solution (pH 1-2) and extracted with
CH2Cl2 (3 ꢀ 10 mL). The combined organic phases were dried
over Na2SO4, then filtered, and concentrated in vacuo. The
crude solid was washed with EtOAc/PE 2:8 to afford 8m
(1.88 g, 98%) as a solid. Mp 105-106 °C (EtOAc/PE). IR
(KBr): ν 3276, 3049, 2962, 1627, 1542, 1344, 1313, 1229, 1092,
1
747 cm-1. H NMR (DMSO-d6): δ 0.98 (t, 3H, J = 7.2 Hz,
CH3), 2.97 (t, 2H, J = 7.0 Hz, CH2), 3.31 (q, 2H, J = 7.2 Hz,
CH2), 3.50 (broad q, 2H, J = 7.0 Hz, CH2), 5.96 (s, 2H, CH2),
6.95-7.00 (m, 1H, Harom), 7.08 (s, 1H, Harom), 7.13 (t, 1H, J =
7.1 Hz, Harom), 7.26-7.34 (m, 3H, Harom), 7.61 (d, 1H, J =
8.3 Hz, Harom), 7.64 (d, 1H, J = 7.9 Hz, Harom), 7.85 (d, 1H, J =
7.9 Hz, Harom), 8.70 (broad t, 1H, J = 5.3 Hz, NH). 13C NMR
(DMSO-d6): δ 14.9 (CH3), 38.9 (CH2), 39.6 (CH2), 63.0
(CH2), 72.5 (CH2), 101.0 (C), 106.1 (CH), 111.2 (CH), 120.8
(CH), 121.6 (CH), 123.9 (CH), 126.0 (C), 128.4 (2 CH),
130.1 (CH), 132.3 (C), 138.3 (C), 139.1 (CH), 141.8 (C), 161.7
(CO). MS (IS): m/z 449 (M þ Hþ). Anal. (C20H21IN2O2)
C, H, N.
tert-Butyl-{[1-(ethoxymethyl)-1H-indol-2-yl]carbonyl}[3-(2-
iodophenyl)propyl]carbamate (9n). According to the procedure
described for the synthesis of 9l, compound 9n was prepared
from 8n in 99% yield as an oil (chromatography eluent: PE/
EtOAc 95:5). IR (film): ν 3063, 2977, 2919, 1732, 1667, 1524,
1
1317, 1144, 1010, 744 cm-1. H NMR (CDCl3): δ 1.15 (t, 3H,
J = 7.2 Hz, CH3), 1.20 (s, 9H, 3 CH3), 1.97-2.07 (m, 2H, CH2),
2.83 (broad t, 2H, J = 7.1 Hz, CH2), 3.56 (q, 2H, J = 7.2 Hz,
CH2), 3.90 (t, 2H, J = 7.1 Hz, CH2), 5.77 (s, 2H, CH2), 6.82 (s,
1H, Harom), 6.86-6.91 (m, 1H, Harom), 7.18 (t, 1H, J = 7.1 Hz,
H
H
H
arom), 7.26-7.28 (m, 2H, Harom), 7.34 (t, 1H, J = 7.9 Hz,
arom), 7.57 (d, 1H, J = 8.5 Hz, Harom), 7.62 (d, 1H, J=7.9 Hz,
arom), 7.81 (d, 1H, J = 8.1 Hz, Harom). 13C NMR (CDCl3): δ
1-(Ethoxymethyl)-N-[3-(2-iodophenyl)propyl]-1H-indole-2-car-
boxamide (8n). According to the procedure described for the
synthesis of 8m, compound 8n was prepared from 4 and 7n in
97% yield. Mp 99-100 °C (EtOAc/PE). IR (KBr): ν 3321, 3045,
14.9 (CH3), 27.5 (3CH3), 29.3 (CH2), 38.2 (CH2), 45.3 (CH2),
63.8 (CH2), 73.4 (CH2), 82.8 (C), 100.4 (C), 108.0 (CH), 110.9
(CH), 121.2 (CH), 121.9 (CH), 124.8 (CH), 126.2 (C), 127.8
(CH), 128.3 (CH), 129.2 (CH), 133.7 (C), 138.6 (C), 139.3 (CH),
143.9 (C), 153.6 (C), 165.7 (CO). MS (IS): m/z 563 (M þ Hþ).
Anal. (C26H31IN2O4) C, H, N.
1
2965, 1638, 1548, 1448, 1310, 1226, 1093, 1009, 751 cm-1. H
NMR (DMSO-d6): δ 0.99 (t, 3H, J = 7.2 Hz, CH3), 1.75-1.85
(m, 2H, CH2), 2.71-2.77 (m, 2H, CH2), 3.28-3.40 (m, 4H, 2
CH2), 5.98 (s, 2H, CH2), 6.93-6.99 (m, 1H, Harom), 7.11 (s, 1H,
1-(Ethoxymethyl)-N-[2-(2-iodophenyl)ethyl]-N-methyl-1H-in-
dole-2-carboxamide (9o). At 0 °C, sodium hydride (27 mg,
0.67 mmol, 60% dispersed in oil) was added to a solution of
9m (200 mg, 0.45 mmol) in THF (1.5 mL). The reaction mixture
was stirred for 10 min at room temperature, and iodomethane
(34 μL, 0.53 mmol) was added dropwise. The mixture was stirred
for 4 h at room temperature. The solvent was evaporated
in vacuo. The residue was taken up in H2O (5 mL) and extracted
with CH2Cl2 (2 ꢀ 5 mL). The combined organic phases were
dried over Na2SO4 and concentrated in vacuo. The crude residue
was purified by flash chromatography (PE/EtOAc 65:35) to give
9o (204 mg, 99%) as an oil. IR (film): ν 3056, 2975, 2936, 1633,
1538, 1455, 1400, 1312, 1180, 1095, 1013, 742 cm-1. 1H NMR
(DMSO-d6 at 75 °C): δ 1.02 (t, 3H, J = 7.0 Hz, CH3), 3.06 (q,
2H, J = 7.0 Hz, CH2), 3.09 (s, 3H, CH3), 3.37 (q, 2H, J = 7.0 Hz,
CH2), 3.70 (broad t, 2H, J = 7.4 Hz, CH2), 5.55 (broad s, 2H,
CH2), 6.58 (broad s, 1H, Harom), 6.96-7.01 (m, 1H, Harom), 7.12 (t,
1H, J = 7.5 Hz, Harom), 7.22-7.37 (m, 3H, Harom), 7.58 (broad d,
2H, J = 9.0 Hz, Harom), 7.83 (d, 1H, J = 7.8 Hz, Harom). MS (IS):
m/z 463 (M þ Hþ). Anal. (C21H23IN2O2) C, H, N.
Harom), 7.13 (t, 1H, J = 7.7 Hz, Harom), 7.28 (t, 1H, J = 8.1 Hz,
Harom), 7.30-7.36 (m, 2H, Harom), 7.61 (d, 1H, J = 8.3 Hz,
Harom), 7.65 (d, 1H, J = 8.3 Hz, Harom), 7.83 (d, 1H, J = 7.7 Hz,
H
arom), 8.65 (t, 1H, J = 5.5 Hz, NH). 13C NMR (DMSO-d6):
δ 14.9 (CH3), 29.8 (CH2), 37.6 (CH2), 38.3 (CH2), 63.0
(CH2), 72.4 (CH2), 100.7 (C), 106.0 (CH), 111.2 (CH), 120.8
(CH), 121.6 (CH), 123.9 (CH), 126.0 (C), 128.1 (CH), 128.6
(CH), 129.7 (CH), 132.5 (C), 138.3 (C), 139.1 (CH), 144.1 (C),
161.7 (CO). MS (IS): m/z 463 (M þ Hþ). Anal. (C21H23IN2O2)
C, H, N.
tert-Butyl-{[1-(ethoxymethyl)-1H-indol-2-yl]carbonyl}(2-iodo-
phenyl)carbamate (9l). A solution of amide 8l (800 mg, 1.9 mmol),
Boc2O (663 mg, 3.04 mmol), and a catalytic amount of DMAP (46
mg, 0.38 mmol) in MeCN (30 mL) was stirred overnight at room
temperature. After evaporation of the solvent, the residue was
partitioned between EtOAc (10 mL) and H2O (10 mL). The two
phases were separated, and the aqueous phase was extracted with
EtOAc (2 ꢀ 10 mL). The combined organic phases were dried over
Na2SO4 and concentrated in vacuo. The crude residue was purified

Article
tert-Butyl-7-(ethoxymethyl)-6-oxo-6,7-dihydroindolo[2,3-c]-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5923
126.4 (CH), 127.0 (C), 127.3 (CH), 129.4 (C), 131.9 (CH), 132.5
(C), 133.1 (CH), 134.6 (C), 138.0 (C), 164.9 (CO). MS (IS): m/z
335 (M þ Hþ). Anal. (C21H22N2O2) C, H, N.
quinoline-5-carboxylate (10l). A mixture of 9l (650 mg, 1.25 mmol),
Pd(OAc)2 (56 mg, 0.25 mmol), PPh3 (131 mg, 0.5 mmol), and
Ag2CO3 (688 mg, 2.50 mmol) in DMF (25 mL) was vigorously
stirred at 100 °C for 1 h. After cooling, the solvent was removed
in vacuo. The residue was taken up in MeOH/CH2Cl2 (9:1), filtered
over Celite, and rinsed with CH2Cl2. The solvent was evaporated
in vacuo and the crude residue was purified by flash chromato-
graphy (PE/EtOAc 85:15 to EtOAc) to give 10l (347 mg, 71%)
as an oil. IR (film): ν 3070, 2977, 1768, 1654, 1466, 1231, 1146,
740 cm-1. 1H NMR (CDCl3): δ 1.12 (t, 3H, J = 7.0 Hz, CH3), 1.75
(s, 9H, 3 CH3), 3.59 (q, 2H, J = 7.0 Hz, CH2), 6.37 (s, 2H, CH2),
7.26-7.28 (m, 1H, Harom), 7.41-7.48 (m, 3H, Harom), 7.58 (t, 1H,
J = 7.8 Hz, Harom), 7.80 (d, 1H, J = 8.3 Hz, Harom), 8.43 (d, 1H,
J = 8.1 Hz, Harom), 8.51 (dd, 1H, J = 2.1, 7.5 Hz, Harom). 13C
NMR (CDCl3): δ 15.2 (CH3), 27.8 (3 CH3), 63.9 (CH2), 73.8
(CH2), 86.8 (C), 112.3 (CH), 114.4 (CH), 118.7 (C), 121.1 (C),
122.2 (CH), 122.5 (C), 122.9 (CH), 123.5 (CH), 124.1 (CH), 125.1
(C), 127.0 (2 CH), 133.4 (C), 140.8 (C), 151.4 (CO), 154.7 (CO).
MS (IS): m/z 415 (M þ Naþ). Anal. (C23H24N2O4) C, H, N.
tert-Butyl-9-(ethoxymethyl)-8-oxo-5,6-dihydro-9H-indolo-
[2,3-e][3]benzazocine-7-carboxylate (10m). According to the pro-
cedure described for the synthesis of 10l (Pd(OAc)2 = 5 mol %,
temperature=140 °C, reaction time=1 h), compound 10m was
prepared from 9m in 97% yield as an oil (chromatography
eluent: PE/EtOAc 85:15). IR (film): ν 3057, 2978, 2936, 1758,
5,7-Dihydroindolo[2,3-c]quinolin-6-one (1l). A solution of 10l
(300 mg, 0.76 mmol) and 1 N HCl (6.2 mL) in 1,4-dioxane
(13 mL) was stirred at 80 °C for 3 h. After cooling, the solution
was neutralized by a saturated aqueous NaHCO3 solution (pH
10) and extracted with CH2Cl2 (2 ꢀ 10 mL). The combined
organic phases were dried over Na2SO4 and concentrated in
vacuo. The crude solid was washed with cold MeOH to afford 1l
(126 mg, 70%). Mp > 210 °C (washing cold MeOH). IR (KBr):
ν 3319, 3156, 3007, 2971, 1660, 1613, 1539, 1329, 1043, 732
cm-1. 1H NMR (DMSO-d6): δ 7.29-7.52 (m, 5H, Harom), 7.64
(d, 1H, J =8.1 Hz, Harom), 8.44-8.50 (m, 2H, Harom), 11.86 (s,
1H, NH), 12.35 (s, 1H, NH). 13C NMR (DMSO-d6): δ 113.1
(CH), 116.2 (CH), 118.1 (C), 118.3 (C), 120.7 (CH), 122.3 (C),
122.4 (2 CH), 123.1 (CH), 125.7 (CH), 126.0 (CH), 127.7 (C),
135.0 (C), 138.9 (C), 155.8 (CO). MS (IS): m/z 235 (MþHþ).
Anal. (C15H10N2O) C, H, N.
5,6,7,9-Tetrahydroindolo[2,3-e][3]benzazocin-8-one (1m). Ac-
cording to the procedure described for the synthesis of 1l
(reaction time = 5 h), compound 1m was prepared from 10m
in 28% yield as a solid. Compound 1m was purified by flash
chromatography (chromatography eluent: CH2Cl2/MeOH
98.5:1.5). Mp > 210 °C (washing cold CH2Cl2). IR (KBr): ν
3255, 3168, 3046, 2922, 1623, 1542, 1460, 1336, 1294, 746 cm-1
.
1716, 1682, 1455, 1368, 1148, 1095, 740 cm-1 1H NMR
.
1H NMR (DMSO-d6 þ D2O at 90 °C): δ 2.97 (t, 2H, J = 6.9 Hz,
(CDCl3): δ 1.17 (t, 3H, J = 7.2 Hz, CH3), 1.23 (s, 9H, 3 CH3),
2.89-2.96 (m, 2H, CH2), 3.51-3.71 (m, 3H, CH2), 4.19-4.29
(m, 1H, CH2), 5.87 (d, 1H, J = 10.7 Hz, CH2), 6.00 (d, 1H, J =
10.7 Hz, CH2), 7.20 (t, 1H, J = 7.4 Hz, Harom), 7.33-7.36 (m,
3H, Harom), 7.41-7.46 (m, 2H, Harom), 7.60-7.63 (m, 2H,
CH2), 3.51 (t, 2H, J=6.9 Hz, CH2), 7.08 (t, 1H, J = 7.0 Hz,
H
H
arom), 7.24 (t, 1H, J = 7.7 Hz, Harom), 7.28-7.52 (m, 6H,
arom). 13C NMR (DMSO-d6): δ 32.8 (CH2), 45.4 (CH2), 112.2
(CH), 114.9 (C), 120.0 (CH), 120.1 (CH), 123.5 (CH), 125.8 (C),
126.3 (CH), 127.0 (CH), 129.9 (C), 130.0 (CH), 130.1 (CH),
133.7 (C), 136,6 (C), 137.5 (C), 166.2 (CO). MS (IS): m/z 263
(M þ Hþ). Anal. (C17H14N2O) C, H, N.
H
arom). 13C NMR (CDCl3): δ 15.0 (CH3), 27.8 (3 CH3), 33.1
(CH2), 47.7 (CH2), 64.1 (CH2), 73.5 (CH2), 81.7 (C), 111.1 (CH),
121.7 (CH), 122.2 (CH), 124.8 (C), 126.5 (C þ 2 CH), 128.0
(CH), 129.5 (C), 130.9 (CH), 131.3 (CH), 133.0 (C), 136.5 (C),
139.8 (C), 151.8 (C), 166.1 (CO). MS (IS): m/z 421 (M þ Hþ).
Anal. (C25H28N2O4) C, H, N.
5,6,7,10-Tetrahydroindolo[2,3-f][4]benzazonin-9(8H)-one (1n).
According to the procedure described for the synthesis of 1l
(reaction time = 1 h), compound 1n was prepared from 10n in
51% yield as a solid. Compound 1n was purified by flash
chromatography (chromatography eluent: CH2Cl2/MeOH
98.5:1.5). Mp > 210 °C (washing cold MeOH). IR (KBr): ν
tert-Butyl-10-(ethoxymethyl)-9-oxo-5,6,7,10-tetrahydroin-
dolo[2,3-f][4]benzazonine-8-carboxylate (10n). According to the
procedure described for the synthesis of 10l, (Pd(OAc)2 =5mol%,
temperature = 140 °C, reaction time = 1 h), compound 10n was
prepared from 9n in 47% yield as an oil (chromatography eluent:
PE/EtOAc 85:15). IR (film): ν 3047, 2969, 2930, 1716, 1693, 1465,
1337, 1297, 1149, 1092, 732 cm-1. 1H NMR (CDCl3): δ 1.16 (s, 9H,
3 CH3), 1.19 (t, 3H, J = 7.2 Hz, CH3), 1.90-1.95 (m, 1H, CH2),
2.11-2.23 (m, 1H, CH2), 2.47-2.57 (m, 1H, CH2), 2.74-2.80
(m, 1H, CH2), 3.48-3.63 (m, 3H, CH2), 3.87 (dd, 1H, J = 4.0, 14.5
Hz, CH2), 5.74 (d, 1H, J= 10.7 Hz, CH2), 5.83 (d, 1H, J= 10.7 Hz,
CH2), 7.14-7.47 (m, 7H, Harom), 7.59 (d, 1H, J = 8.5 Hz, Harom).
13C NMR (CDCl3): δ 15.0 (CH3), 27.5 (CH2), 27.7 (3 CH3),
31.2 (CH2), 46.1 (CH2), 64.1 (CH2), 73.4 (CH2), 81.5 (C), 110.5
(CH), 121.3 (CH), 121.8 (CH), 122.9 (C), 125.2 (CH), 125.6 (CH),
127.1 (C), 128.0 (CH), 129.2 (CH), 130.9 (C), 131.2 (CH), 131.9
(C), 138.5 (C), 141.9 (C), 152.1 (C), 167.2 (CO). MS (IS):
m/z 435 (M þ Hþ). Anal. (C26H30N2O4) C, H, N.
3279, 3227, 3057, 2930, 1658, 1634, 1561, 1408, 1332, 747 cm-1
.
1H NMR (DMSO-d6 þ D2O): δ 1.58 (broad s, 1H, CH2), 1.83
(broad s, 1H, CH2), 2.13 (broad s, 1H, CH2), 2.73 (broad s, 1H,
CH2), 3.00 (broad s, 1H, CH2), 3.25 (broad s, 1H, CH2),
7.00-7.05 (m, 2H, Harom), 7.17-7.22 (m, 3H, Harom), 7.30-
7.31 (m, 2H, Harom), 7.46 (d, 1H, J = 8.1 Hz, Harom). 13C NMR
(DMSO-d6): δ 32.7 (CH2), 34.0 (CH2), 44.2 (CH2), 111.8 (CH),
114.5 (C), 119.3 (CH), 119.7 (CH), 122.5 (CH), 125.3 (CH),
127.1 (CH), 127.8 (C), 129.6 (CH), 131.3 (CH), 131.7 (CH),
132.9 (C), 135.4 (C), 143.1 (C), 166.5 (CO). MS (IS) m/z 277
(M þ Hþ). Anal. (C18H16N2O) C, H, N.
7-Methyl-5,6,7,9-tetrahydroindolo[2,3-e][3]benzazocin-8-one
(1o). According to the procedure described for the synthesis of 1l
(reaction time = 2 h), compound 1o was prepared from 10o in
67% yield as a solid. Compound 1o was purified by flash
chromatography (chromatography eluent: PE/EtOAc 1:1).
Mp > 210 °C (washing cold CH2Cl2). IR (KBr): ν 3215, 3070,
9-(Ethoxymethyl)-7-methyl-5,6,7,9-tetrahydro-8H-indolo[2,3-
e][3]benzazocin-8-one (10o). According to the procedure de-
scribed for the synthesis of 10l, (Pd(OAc)2 = 5 mol %, tem-
perature = 140 °C, reaction time = 3.5 h), compound 10o was
prepared from 9o in 87% yield as an oil (chromatography
eluent: PE/EtOAc 3:2). IR (film): ν 3056, 2971, 1640, 1542,
1454, 1329, 1219, 1099, 755 cm-1. 1H NMR (CDCl3): δ 1.15 (t,
3H. J = 7.0 Hz, CH3), 3.10 (s, 3H, CH3), 3.18-3.34 (m, 3H,
CH2), 3.42-3.57 (m, 2H, CH2), 4.26-4.37 (m, 1H, CH2), 5.63
(d, 1H, J = 10.9 Hz, CH2), 5.84 (d, 1H, J = 10.9 Hz, CH2),
7.18-7.38 (m, 6H, Harom), 7.59 (d, 1H, J = 8.3 Hz, Harom), 7.64
(d, 1H, J = 7.9 Hz, Harom). 13C NMR (CDCl3): δ 15.0 (CH3),
32.7 (CH3), 34.0 (CH2), 50.8 (CH2), 64.0 (CH2), 73.5 (CH2),
110.6 (CH), 118.2 (C), 120.8 (CH), 121.2 (CH), 124.0 (CH),
1
2992, 1613, 1548, 1505, 1404, 1326, 1259, 1085, 745 cm-1. H
NMR (DMSO-d6): δ 2.96 (s, 3H, CH3), 3.18 (s, 2H, CH2), 3.72
(broad s, 2H, CH2), 7.09 (t, 1H, J = 7.4 Hz, Harom), 7.20-7.31
(m, 5H, Harom), 7.45 (d, 1H, J = 8.1 Hz, Harom), 7.52 (d, 1H, J =
7.9 Hz, Harom), 11.78 (s, 1H, NH). 13C NMR (DMSO-d6): δ 33.2
(CH3), 33.3 (CH2), 51.3 (CH2), 112.2 (CH), 114.9 (C), 119.9
(CH), 120.1 (CH), 123.1 (CH), 126.1 (C), 126.2 (CH), 126.9
(CH), 128.9 (C), 131.8 (CH), 131.9 (CH), 132.6 (C), 135.5 (C),
136.4 (C), 164.8 (CO). MS (IS): m/z 277 (M þ Hþ). Anal.
(C18H16N2O) C, H, N.
Biology. Reagents. Paclitaxel was obtained from Bristol-
Myers Squibb (Paris, France). 5-FU, propidium iodide, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),

5924 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Putey et al.
Hoescht 33258, vinblastine, vinorelbine, and colchicine (Bio-
chemika, g97.0%, HPLC) were purchased from Sigma.
Cell Lines. All cell lines were grown on 25 cm2 flasks and were
placed at 37 °C in a humidified atmosphere containing 5% CO2.
The MCF7 and the UACC812 breast cancer cell lines and the
HCT116, SW48, and SW480 colorectal cancer cell lines were
cultured in DMEM culture medium containing 10% fetal calf
serum, 1% L-glutamine, and 2% penicillin-streptomycin. The
Mes-sa sarcoma cell line and the CEM and RL lymphoma cell
lines were cultured in RPMI1640 culture medium contain-
ing 10% fetal calf serum, 1% L-glutamine, and 1% penicillin-
streptomycin.
Colchicine-Binding Site Assay. Competition assays were per-
formed as described previously with some modifications.22,23
Five microliters of 2 mM stock solution of different tubulin-
binding drugs to a final concentration of 50 μM were incubated
with 100 μL of tubulin (1.1 mg/mL, 10 μM) at 37 °C for 30 min.
Unlabeled colchicine and vinblastine (not-competitive) were
used as positive and negative controls, respectively. The mixture
was then incubated with fluorescent colchicine at 0.5 μM
(Genolite Biotek, USA) under the same conditions. After
incubation, each reaction was applied to a 5 mL HiTrap desalt-
ing column (GE Healthcare) previously calibrated with compe-
tition binding assay buffer (0.25 mM PIPES, pH 6.9, 0.05 mM
GTP, and 0.25 mM MgCl2) and eluted using 0.25 mL fractions.
The peak position for tubulin was determined with an AKTA
purifier (GE Healthcare) using UV absorbance at 280 nm. Since
GTP also absorbs at this wavelength, the protein peak was
further validated by measuring the protein concentration of the
peak fractions using the Bradford reagent.24 From each frac-
tion, 50 μL were transferred into a 96-well flat bottom poly-
styrene NBS microplate (Product No3650, Corning), and the
fluorescence intensity was measured at an excitation of 493 nm
and an emission wavelength of 516 nm using a TECAN Safire
plate reader. The fluorescence intensity was plotted against the
elution volume. All experiments were at least performed in
triplicate.
Cell Growth Inhibition Assay. Antiproliferative activity was
determined in three separate experiments, each of which was
performed in triplicate as previously described.20 Briefly, asyn-
chronously growing cells were transferred into 96-well culture
plates (Costar, Corning Inc., New York) in 100 μL of medium at
a final concentration of 5 ꢀ 103 cells/well and incubated for 24 h.
Corresponding drug concentrations were then added to each
plate. After 72 h of drug exposure, 20 μL of MTT reagent (5 mg/
mL) was added to each well. Cell growth was expressed as the
percent of absorbance of treated wells relative to the untreated
control wells. IC50 values were defined as drug doses resulting in
50% cell growth inhibition relative to untreated cells.
Cell Cycle Assay. For analysis of DNA content and cell cycle
distribution, colorectal cancer cell lines were treated with com-
pounds 1a, 1b, 1e, and 1m for 24 h. On the basis of the
cytotoxicity assay, a concentration of 30 μM (aprox. IC80 values
for these cell lines) was chosen for drug exposure experiments.
After drug exposure, 106 cells/mL were resuspended in 2 mL of
propidium iodide solution (50 μL/mL), incubated at 4 °C over-
night, and then analyzed by flow cytometry. Flow cytometry
was performed on FACScalibur (Becton Dickinson, San Jose,
California). Cell cycle distribution and DNA ploidy status were
calculated after exclusion of cell doublets and aggregates on a
FL2-area/FL2-width dot plot using Modfit LT 2.0 software
(Verity Software Inc. Topsham. ME).
Immunofluorescence and Microscopy. Exponentially growing
cells were plated on 18-mm microscope glass slides and incu-
bated with 1a, 1b, 1e, and 1m at 10 μM for 24 h. Then, cells were
fixed in 100% methanol. DNA was counterstained with 5 μg/
mL Hoescht 33258. Coverslips were examined with a Zeiss
axioplasm microscope using a Zeiss x100 1.3 oil-immersion
objective.
Purification of Tubulin and MT Polymerization Assays. Tu-
bulin was purified from bovine brain from freshly slaughtered
animals as described earlier.21 In total, we obtained 244 mg of
pure tubulin (Figure S1, Supporting Information) from two
brains. The mixing of tubulin samples with different inhibitor
concentrations was carried out on ice, and only freshly thawed
tubulin was used. Unused tubulin was discarded after 20 min.
Tubulin polymerization experiments were carried out at 37 °C in
a 96-well Sunrise spectrophotometer (TECAN) in 96-well mi-
croclear plates (Greiner BIO-One) at a wavelength of 340 nm.
First, we determined the optimal polymerization conditions by
varying GTP, DMSO, and tubulin concentrations, and the best
conditions were found to be 40 μM tubulin (based on the
concentration of the R,β-tubulin heterodimer), 1.25 mM GTP,
and 2.5% DMSO. Subsequently, tubulin polymerization assays
under these conditions were performed in the absence of in-
hibitors (polymerization control) and in the presence of increas-
ing inhibitor concentrations (1 μM to 75 μM). All compounds
were measured at least in triplicate. Colchicine as an inhibitor of
tubulin polymerization served as additional control. An exam-
ple of the inhibition of tubulin polymerization by compound 1e,
a potent inhibitor analogue in vitro and in cell-based assays,
and 1h, as an example for an inhibitor that does not inhibit
tubulin polymerization, and their corresponding concentra-
tion-response plots are shown in Supporting Information
(Figure S2).
Acknowledgment. We are grateful to Dr. Richard Mac-
Culloch for providing us with fresh bovine brains for tubulin
purification. A. P. was supported by a fellowship from the
ꢁ
“Ministere de l’Education Superieure et de la Recherche”.
ꢀ
Supporting Information Available: Elemental analyses, ex-
perimental procedures, and spectral data of 6, 7, 11, 12, 13, 14,
1g-1s, SDS-PAGE to estimate the purity of the protein, and
polymerization of tubulin in the presence of inhibitors. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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