Synthesis and biological evaluation of 1-methyl-2-(3′,4′, 5′-trimethoxybenzoyl)-3-aminoindoles as a new class of antimitotic agents and tubulin inhibitors

Article abstract of DOI:10.1021/jm7011547

The 2-(3,4,5-trimethoxybenzoyl)-2-aminoindole nucleus was used as the fundamental structure for the synthesis of compounds modified with respect to positions C-4 to C-7 with different moieties (chloro, methyl, or methoxy). Additional structural variations concerned the indole nitrogen, which was alkylated with small alkyl groups such as methyl or ethyl. We have identified 1-methyl-2-(3,4,5-trimethoxybenzoyl)-3-amino-7-methoxyindole as a new highly potent antiproliferative agent that targets tubulin at the colchicine binding site and leads to apoptotic cell death.

Full text of DOI:10.1021/jm7011547

1464
J. Med. Chem. 2008, 51, 1464–1468
Synthesis and Biological Evaluation of 1-Methyl-2-(3′,4′,5′-trimethoxybenzoyl)-3-aminoindoles as
a New Class of Antimitotic Agents and Tubulin Inhibitors
Romeo Romagnoli,*,† Pier Giovanni Baraldi,*,† Taradas Sarkar,‡ Maria Dora Carrion,† Carlota Lopez Cara,† Olga Cruz-Lopez,†
Delia Preti,† Mojgan Aghazadeh Tabrizi,† Manlio Tolomeo,# Stefania Grimaudo,# Antonella Di Cristina,# Nicola Zonta,§
Jan Balzarini, Andrea Brancale,§ Hsing-Pang Hsieh,| and Ernest Hamel‡
Dipartimento di Scienze Farmaceutiche, UniVersità di Ferrara, 44100 Ferrara, Italy, Dipartimento di Oncologia, Policlinico “P. Giaccone”,
DiVisione di Ematologia e SerVizio AIDS, UniVersità di Palermo, Palermo, Italy, Laboratory of Virology and Chemotherapy, Rega Institute for
Medical Research, Minderbroedersstraat 10, B-3000 LeuVen, Belgium, The Welsh School of Pharmacy, Cardiff UniVersity, King Edward VII
AVenue, Cardiff, CF10 3XF, U.K., DiVision of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli,
Taiwan, Republic of China, and DeVelopmental Therapeutics Program, Toxicology and Pharmacology Branch, DiVision of Cancer Treatment
and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702
ReceiVed September 15, 2007
The 2-(3,4,5-trimethoxybenzoyl)-2-aminoindole nucleus was used as the fundamental structure for the
synthesis of compounds modified with respect to positions C-4 to C-7 with different moieties (chloro, methyl,
or methoxy). Additional structural variations concerned the indole nitrogen, which was alkylated with small
alkyl groups such as methyl or ethyl. We have identified 1-methyl-2-(3,4,5-trimethoxybenzoyl)-3-amino-
7-methoxyindole as a new highly potent antiproliferative agent that targets tubulin at the colchicine binding
site and leads to apoptotic cell death.
Chart 1. Inhibitors of Tubulin Polymerization
Introduction
The discovery of natural and synthetic compounds targeting
the mitotic spindle apparatus has attracted much attention with-
in the past 2 decades, and microtubules are recognized as an
important target for the development of potential new chemo-
therapeutic agents.1 More recently, it has been established that
some tubulin-binding agents selectively target tumor vasculature,
and thus, these compounds can also be considered vascular
disrupting agents.2
One of the most important tubulin-binding agents is com-
bretastatin A-4 (CA-4, 1; Chart 1). CA-4, isolated from the bark
of the South African tree Combretum caffrum,3 is one of the
well-known natural tubulin-binding molecules affecting micro-
tubule dynamics. CA-4 strongly inhibits the polymerization of
tubulin by binding to the colchicine site.4 As a result of its
simple chemical structure, many analogues that mimic CA-4
activity have been developed and evaluated in SAR studies.5
Among synthetic small-molecule tubulin inhibitors, replacement
of the double bond of 1 by a carbonyl group furnished a
benzophenone-type CA-4 analogue named phenstatin (2). This
compound demonstrated interesting efficacy in a variety of
tumor models while retaining the characteristics of 1.6 The
2-aminobenzophenone derivative 3 also strongly inhibites cancer
cell growth and tubulin polymerization, and it causes mitotic
arrest, as does 2.7
In a recent study, we reported the discovery of a series of
molecules with general structure 4, characterized by the presence
of a 2-(3,4,5-trimethoxybenzoyl)-3-aminobenzo[b]thiophene skel-
eton, which was designed by bioisosteric replacement of the
* To whom correspondence should be addressed. For R.R.: phone, 39-
(0)532-291290; fax, 39-(0)532-291296; e-mail, rmr@unife.it. For P.G.B.:
phone, 39-(0)532-291293; fax, 39-(0)532-291296; e-mail: baraldi@unife.it.
†
Università di Ferrara.
National Cancer Institute at Frederick.
Università di Palermo.
Cardiff University.
‡
#
§
2-aminobenzene moiety of 3 with a 3-aminobenzo[b]thiophene.
This new class of compounds exerts strong inhibition of tubulin
Rega Institute for Medical Research.
National Health Research Institutes.
|
10.1021/jm7011547 CCC: $40.75
2008 American Chemical Society
Published on Web 02/09/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1465
Scheme 1a
polymerization by binding to the colchicine binding site of tubulin,
and they cause G2/M phase arrest of the cell cycle.8
In this article we are interested in determining if the classical
divalent ring-equivalent bioisosterism,9 exemplified by replace-
ment of the sulfur of the benzo[b]thiophene nucleus of 4 with
a nonsterically hindered NH group, would lead to a novel and
active series of 2-(3,4,5-trimethoxybenzoyl)-3-aminoindole de-
rivatives (compounds 5a-l). By the synthesis of compounds
5b-l, we were able to analyze the effect on biological activity
of electron-donating (methyl and methoxy) and electron-
withdrawing (chloro) substituents at positions 4-7 of the indole
ring. Finally, through the synthesis of compounds 6a-k and
6l-n, we investigated whether the introduction of a methyl and
ethyl moiety, respectively, at the indolic nitrogen of derivatives
5a-l would lead to loss of activity at the colchicine site through
a steric effect.
Antitubulin agents having an indole as their core nucleus have
been recently reviewed,10 and in the past few years an ever
increasing number of synthetic indoles as potent tubulin
polymerization inhibitors have been reported.11 The 2- and
3-aroylindole moiety constituted the core structure of two classes
of antimitotic compounds, with general structures 7 and 8,
respectively.12
Chemistry
The N-1H- and N-1-methyl- or ethyl-3-aminoindoles (com-
pounds 5a-l and 6a-n, respectively) were prepared following
the reaction sequence reported in Scheme 1. 2-Aminobenzoni-
triles 9a-l were converted to the corresponding N-ethoxycar-
bonylanilines 10a-l by treatment with neat ethyl chloroformate
(ClCO2Et) at reflux. The subsequent condensation, followed by
intramolecular cyclization, with (3,4,5-trimethoxyphenyl)-2-
bromoethanone8 using NaH as base in DMF provided the N-1-
ethoxycarbonyl-3-aminoindoles intermediates in good yield.
These were subsequently N-deprotected by alkaline hydrolysis
using NaOH in aqueous ethanol to furnish the corresponding
N-1H-2-(3′,4′,5′-trimethoxybenzoyl)-3-aminoindoles 5a-l. With
the exception of 5k, these latter compounds were transformed
almost quantitatively into the corresponding N-phthalimido
derivatives 11a-k using phthalic anhydride in refluxing acetic
acid. N-1 alkylation of the indole nucleus was easily achieved
by treatment of 11a-k with NaH in DMF, followed by
condensation with methyl or ethyl iodide, to furnish intermedi-
ates 12a-k or 12l-n, respectively. The removal of the
N-protected phthaloyl group was accomplished with hydrazine
in refluxing ethanol to afford final N-1-alkylated 2-(3′,4′,5′-
trimethoxybenzoyl)-3-aminoindoles 6a-n.
a Reagents: (a) ClCO2Et, reflux, 6 h; (b) (3,4,5-trimethoxyphenyl)-2-
bromoethanone, NaH, DMF, room temp, 24 h; (c) NaOH, EtOH/H2O, reflux,
1 h; (d) phthalic anhydride, AcOH; (e) MeI or EtI, NaH, DMF, room temp;
(f) NH2NH2, EtOH.
N-1 position of the indole nucleus, along with its C4–C6
substituent pattern, plays an essential role in the antiproliferative
activity of derivatives. With the exception of C-4 methyl and
methoxy derivatives (compounds 5–6b and 5–6f, respectively),
which proved to be inactive, the N-methylindole derivatives
6a-n showed increased antiproliferative activity in comparison
with their parent N-unsubstituted counterparts 5a-l.
In the N-methylindole series, a comparison of substituent
effects revealed that the antiproliferative effects of the C-6 and
C-7 methoxy compounds 6h and 6i exceeded that of their methyl
counterparts 6d and 6e, respectively, by 2 orders of magnitude.
We observed a different effect at the C-5 position, where the
substitution of a methoxy with a methyl group (compounds 6g
and 6c, respectively) caused only a modest 2- to 3-fold increase
in activity.
In the series of N-H indole derivatives 5b-e, the C-4 (5b),
C-5 (5c), and C-7 (5e) methyl isomers were inactive compounds
(IC50 > 10 µM), while the C-6 methyl substituted derivative
5d showed low antiproliferative activity (IC50 ) 1.4–10 µM).
In the corresponding N-methyl series 6b-e, the C-4 methyl
derivative (6b) was inactive, while the C-5 (6c), C-6 (6d), and
C-7 (6e) isomers had similar activities against L1210 and FM3A
cells, while 6d was more potent than 6c and 6e against Molt4
and CEM cells.
Concerning methoxy substituents, the differences between
C-4/5 versus C-6/7 were even more dramatic with the N-1-H
and N-1-methyl series (5f-i and 6f-i, respectively). For the
former series, the C-4 (5f) and C-5 (5g) methoxy derivatives
Results and Discussion
Table 1 summarizes the growth inhibitory effects of indole
derivatives 5a-l and 6a-n against murine leukemia (L1210),
murine mammary carcinoma (FM3A), and human T-lympho-
blastoid (Molt/4 and CEM) cells, with CA-4 (1), 4a, and 8a as
reference compounds. In general, the antiproliferative activities
of the compounds were greater against the Molt/4 and CEM
cells compared with the two murine cell lines. The most active
compound identified in this study was 6i, which inhibited the
growth of L1210, FM3A, Molt/4, and CEM cancer cell lines
with IC50 values at 15, 15, 3.8, and 8.8 nM, respectively, The
antiproliferative activity of 6i was in the same range as those
of reference compounds 1, 4a, and 8a.
While the unsubstituted indole 5a and its N-1-ethyl derivative
6l proved inactive, a modest improvement in activity (IC50
2–10 µM) was observed for the N-1-methyl analogue 6a. The
)

1466 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Brief Articles
Table 1. In Vitro Inhibitory Effects of Compounds 4a, 5a-l, 6a-n, 8a,
and CA-4 (1) on the Proliferation of Murine Leukemia (L1210), Murine
Mammary Carcinoma (FM3A), and Human T-Lymphocyte (Molt/4 and
CEM) Cells
Table 2. Inhibition of Tubulin Polymerization and Colchicine Binding
by Compounds 4a, 5h, 6c-e,g-k, 8a, and CA-4
tubulin assembly,a
colchicine binding,b
compd
5h
6c
6d
6e
6g
6h
6i
6j
6k
4a
8a
CA-4(1)
IC50 ( SD (µM)
% inhibition ( SD
IC50 (nM)a
>40
nd
nd
nd
nd
10 ( 0.2
6.4 ( 0.07
8.4 ( 0.1
15 ( 2
compd
5a
5b
5c
5d
5e
5f
L1210
FM3A
Molt4/C8
CEM
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
nd
>10000
>10000
>10000
>10000
4.6 ( 0.07
1.4 ( 0.07
18 ( 1
7.1 ( 0.3
2.1 ( 0.3
2.9 ( 0.1
1.2 ( 0.1
38 ( 5
76 ( 5
nd
3500 ( 150
>10000
>10000
1400 ( 80
>10000
2600 ( 0
>10000
>10000
>10000
>10000
>10000
>10000
nd
5g
5h
5i
5j
5k
5l
6a
6b
6c
6d
6e
9700 ( 0
970 ( 24
>10000
9900 ( 30
1600 ( 30
>10000
6900 ( 190
630 ( 10
640 ( 260
8600 ( 110
>10000
9200 ( 60
2100 ( 0.0
>10000
1700 ( 100
110 ( 30
270 ( 90
>10000
8600 ( 30
320 ( 90
630 ( 10
9500 ( 0
>10000
39 ( 1
84 ( 0.7
86 ( 3
>10000
>10000
a Inhibition of tubulin polymerization. Tubulin was at 10 µM. b Inhibition
of [3H]colchicine binding. Tubulin, colchicine, and tested compound were
at 1, 5, and 5 µM, respectively. nd: not determined.
>10000
>10000
>10000
>10000
>10000
8100 ( 37
>10000
1900 ( 300
1900 ( 150
1800 ( 120
>10000
>10000
6800 ( 80
>10000
1700 ( 200
400 ( 110
1200 ( 400
>10000
>10000
1600 ( 200
1500 ( 90
1400 ( 110
>10000
5h, which did not inhibit tubulin assembly at a concentration
as high as 40 µM, there was a positive correlation between the
inhibition of tubulin polymerization and antiproliferative activity.
The most potent compound in this series was 6i (IC50 ) 1.4
µM), ∼2 times more active than 4a and 8a (2.1 and 2.9 µM,
respectively) and about as active as 1.
Colchicine binding studies were performed on compounds
(6d and 6i) with tubulin assembly IC50 of <5 µM. Compound
6i potently inhibited the binding of [3H]colchicine to tubulin,
with 76% inhibition occurring when 6i and radiolabeled
colchicine were equimolar (5 µM each) in the reaction mixture.
This derivative was slightly less potent than CA-4 and 8a, which
in these experiments inhibited colchicine binding by 86% and
84%, respectively, but 6i was 2-fold more potent than its
benzo[b]thiophene counterpart 4a.
6f
6g
6h
6i
6j
6k
6l
6m
6n
4a
8a
CA-4(1)
4200 ( 110
69 ( 37
15 ( 4
5400 ( 270
1100 ( 60
>10000
>10000
>10000
33 ( 29
3.5 ( 1
2.8 ( 1.1
5200 ( 90
97 ( 3
2100 ( 0
57 ( 7
3.8 ( 0.6
5000 ( 440
480 ( 0
>10000
>10000
9100 ( 210
8.5 ( 1.4
3.4 ( 0.0
1.6 ( 1.4
3900 ( 100
71 ( 5
15 ( 0
8.8 ( 1
5000 ( 430
970 ( 75
>10000
>10000
>10000
27 ( 13
3.7 ( 0.2
42 ( 6
5000 ( 260
1000 ( 60
>10000
>10000
>10000
8.9 ( 2.0
3.6 ( 1
1.9 ( 1.6
a IC50: compound concentration required to inhibit tumor cell proliferation
by 50%. Data are expressed as the mean ( SE from the dose–response
curves of at least three independent experiments.
Because molecules exhibiting effects on tubulin assembly
should cause alteration of cell cycle parameters, with preferential
G2-M blockade, flow cytometry analysis was performed to
determine the effect of the most active compounds on K562
(human chronic myelogenous leukemia) cells. Cells were
cultured for 24 h in the presence of each compound at the IC50
determined after 24 h of growth (5h, 3 µM; 6h, 200 nM; 6i, 30
nM).
Compounds 5h and 6h-i caused a marked increase in the
percentage of cells blocked in the G2-M phase of the cell cycle,
with a simultaneous decrease of cells in the S and G0-G1 phases
(Figure 1). Of interest, 24 h exposure of K562 cells with 5h
and 6h,i used at the above concentrations induced about 25%
apoptosis (sub-G0-G1 peak). After 48 h of treatment, 6i showed
an AC50 (concentration able to induce apoptosis in 50% of cells)
of 30 nM. When used at 60 nM, 6i was able to kill by apoptosis
100% of the K562 cells. In terms of the AC50 values, 6i was 5
times more active than 6h (AC50 ) 150 nM) and 100 times
more active than 5h (AC50 ) 3 µM).
Molecular docking experiments were performed on the series
of compounds reported to identify a possible binding mode for
these compounds, using MOE15 and following the same
procedure reported before.8 Compound 6i appears to bind in a
very similar manner to colchicine (Figure 2). The trimethoxy-
phenyl moiety lies in proximity to ꢀCys241 (the residue
numbering is based on the crystal structure used16), and the
methoxy group occupies the same position as the corresponding
group on ring C of colchicine. Furthermore, the N-methyl moiety
forms a tight hydrophobic contact with ꢀLeu255 and ꢀMet259.
This could explain the observed experimental results for 6i and
the loss of biological activity for compounds 5i and 6n. The
ethyl group of 6n is too big to be accommodated in the same
were inactive, while a 6- to 20-fold increase in potency was
observed by moving the methoxy from the C-5 (5g) to the C-6
(5h) position. A loss of activity against L1210 and FM3A cells
was observed for the C-7 derivative 5i.
In N-1-methyl compounds 6f-i, the greatest activity occurred
when the methoxy group was introduced at the C-6 or C-7
position, the least when located at the C-4 or C-5 position. For
the compounds 6f-i, the C-6 (6h) and C-7 (6i) derivatives were
the most active compounds of the whole series and displayed
inhibitory activities comparable to that of CA-4 against four
cancer cell lines. The potency improvement by adding a methyl
group at the N-1-position intrigued us and caused us to explore
whether the N-1-ethyl series exhibited a similar effect.
From a comparison of the data for compounds 6h,i with those
of 6m,n, with an ethyl group on the indolic nitrogen, it is clear
that extension of the alkyl chain by an extra methylene unit
results in a drastic reduction in activity. There thus appears to
be a substantial steric effect resulting from substitution at the
N-1 position of the indole. A considerable loss of cytostatic
activity was observed with methoxy substituents at C-5 and C-6
(5–6j).
By comparing the effect of substituents with opposite
electronic properties at the C-6 position of the indole nucleus,
we found that replacement of the electron-donating methoxy
(5–6h) group with an electron-withdrawing chlorine atom (5–6k)
strongly decreased growth inhibitory properties.
To investigate whether the antiproliferative activities of these
compounds were related to an interaction with the microtubule
system, 5h and 6c-e,g-k were evaluated for their inhibitory
effects on tubulin polymerization and on the binding of
[3H]colchicine to tubulin (Table 2).13,14 With the exception of

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1467
Figure 2. Proposed binding mode of 6i. DAMA-colchicine is repre-
sented in red.
proposed binding mode for 6i in the colchicine site of tubulin
is consistent with the experimental data.
Figure 1. Effects of compounds 5h (b), 6h (c), and 6i (d) on DNA
content/cell following treatment of K562 cells for 24 h. The cells were
cultured without any compound (a) or with compound used at the
concentration leading to 50% cell growth inhibition after 24 h of
treatment. Cell cycle distribution was analyzed by the standard
propidium iodide procedure as described in the Experimental Section
of Supporting Information. Sub-G0-G1 (A), G0-G1, S, and G2-M cells
without treatment are indicated in panel a.
Experimental Section
General Procedure A for the Synthesis of Compounds 10a-l.
A vigorously stirred suspension of 2-aminoarylnitriles 8a-l (5
mmol) in ethyl chloroformate (2.5 mL, 25 mmol) was heated to
reflux for 6 h. After complete removal of excess ethyl chloroformate
in vacuo, the residue recrystallized from petroleum ether furnished
the title compound, which was used without purification for the
next reaction.
General Procedure B for the Synthesis of 2-(3′,4′,5′-Tri-
methoxybenzoyl)-N-ethoxycarbonylindoles. Sodium hydride (96
mg, 50% dispersion in mineral oil, 2 mmol) was added to a solution
of 2-N-ethoxycarbonylarylnitriles 9a-l (2 mmol) in DMF (5 mL),
and the mixture was stirred for 1 h. Then a solution of 2-bromo-
1-(3,4,5-trimethoxyphenyl)ethanone (580 mg, 2 mmol) was added
in small portions, and the mixture was stirred at room temperature
for 18 h. The mixture was poured into water (10 mL) and extracted
with dichloromethane (3 × 20 mL). The combined extracts were
washed with brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by flash column chromatography on silica
gel to afford the title compounds.
position as the methyl group, while the loss of the hydrophobic
interaction would reduce the binding affinity for the N unsub-
stituted 5i. It should be noted that 6n and 5i did not dock in a
similar fashion as 6i. Docking of 6n did not generate any pose
with the trimethoxy ring positioned within 3 Å of the corre-
sponding ring A of colchicine, while the best scored pose for
5i, despite a good overlap between the trimethoxyphenyl ring
and the colchicine ring A, placed the indole ring almost outside
the binding pocket, indicating less efficient binding (see
Supporting Information).
Conclusions
In conclusion, the synthesis and biological evaluation of a
new class of synthetic antitubulin compounds based on the
2-(3′,4′,5′-trimethoxybenzoyl)-3-aminoindole skeleton are de-
scribed. We have demonstrated that the presence of a hydrogen
on the indole nitrogen was detrimental for antiproliferative
activity, while N-methyl alkylation plays a decisive role
increasing antiproliferative potency.
Many active compounds were found among the 1-methyl-2-
(3,4,5-trimethoxybenzoyl)-3-aminoindole series, with the C-6
and C-7 methoxy derivatives 6h and 6i being the most active,
with antiproliferative IC50 values ranging from 57 to 97 and
3.8–15 nM, respectively, against the four cancer cell lines.
Compound 6i was both the most active antiproliferative agent
and the most effective inhibitor of tubulin polymerization among
the newly synthesized compounds. Its activities closely parallel
those of reference compounds CA-4 and 8a. 6i was more active
than the corresponding benzo[b]thiophene analogue 4a as an
inhibitor of cancer cell growth and tubulin polymerization.
We also showed by flow cytometry that 6i had cellular effects
typical of agents that bind to tubulin, causing accumulation of
cells in the G2-M phase of the cell cycle and extensive apoptosis.
Molecular modeling studies were also performed, and the
General Procedure C for the Synthesis of Compounds
5a-l. A solution of NaOH (400 mg, 10 mmol) in water (2 mL)
was added to a stirred solution of 2-(3′,4′,5′-trimethoxybenzoyl)-
N-ethoxycarbonylindoles (1 mmol) in EtOH (3 mL), and the mixture
was refluxed for 15 min. The mixture was evaporated to dryness,
and water (5 mL) was added to the residue. After acidification of
the resulting solution with 0.1 N aqueous solution HCl (pH 3–4)
and extraction with dichloromethane (3 × 10 mL), the combined
extracts were washed with brine and dried over Na2SO4. The residue
was purified by flash column chromatography on silica gel to afford
the title compounds (EtOAc-petroleum ether, 3:7, for 5b-d,
EtOAc-petroleum ether, 1:1, for 5a,e-l).
General Procedure D for the Synthesis of Compounds
11a-k. To a suspension of 5a-j or 5l (6 mmol) in acetic acid (20
mL) was added phthalic anhydride (7.2 mmol, 1.07 g). After the
mixture was stirred for 18 h at reflux, the solvent was evaporated
and the residue dissolved in EtOAc (30 mL). The organic solution
was washed with a saturated solution of NaHCO3 (10 mL), water
(10 mL), and brine (10 mL), dried, and concentrated. The crude
product was purified by crystallization from petroleum ether.
General Procedure E for the Synthesis of Compounds
12a-n. Sodium hydride (60% oil dispersion, 40 mg, 1 mmol, 1
equiv) was carefully added to a solution of alkyl iodide (3 mmol,
3 equiv) and indole derivative 11a-k (1 mmol) in 5 mL of dry

1468 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Brief Articles
(8) Romagnoli, R.; Baraldi, P. G.; Carrion, M. D.; Lopez Cara, C.; Preti,
D.; Fruttarolo, F.; Pavani, M. G.; Tabrizi, M. A.; Tolomeo, M.;
Grimaudo, S.; Di Antonella, C.; Balzarini, J.; Hadfield, J. A.; Brancale,
A.; Hamel, E. Synthesis and biological evaluation of 2- and 3-ami-
nobenzo[b]thiophene derivatives as antimitotic agents and inhibitors
of tubulin polymerization. J. Med. Chem. 2007, 50, 2273–2277.
(9) Lima, L. M.; Barreiro, E. J. Bioisosterism: a useful strategy for
molecular modification and drug design. Curr. Med. Chem. 2005, 12
(1), 23–49.
DMF at 0 °C. The mixture was allowed to warm to room
temperature and was stirred for 2 h. The mixture was poured into
water (10 mL) and extracted with dichloromethane (3 × 10 mL).
The combined organic extracts were washed with water (3 × 10
mL) and brine, dried, and concentrated in vacuo. The residue was
purified by column chromatography.
General Procedure F for the Synthesis of Compounds
6a-n. A stirred suspension of indole derivative (12a-n, 0.5 mmol)
and hydrazine monohydrate (29 µL, 0.6 mmol, 1.2 equiv) in
absolute EtOH (10 mL) was refluxed for 3 h. The solvent was
evaporated, and the residue was partitioned between EtOAc (10
mL) and water (5 mL). The separated organic phase, washed with
brine (2 mL) and dried, was concentrated under vacuo to obtain a
residue that was purified by column chromatography to afford the
title compounds (EtOAc-petroleum ether, 6:4, for 6a,b,d,e,
h-j,l-n; EtOAc-petroleum ether, 1:1, for 6c,f,g,k).
(10) Brancale, A.; Silvestri, R. Indole, a core nucleus for potent inhibitors
of tubulin polymerization. Med. Res. ReV. 2006, 27, 209–238, and
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Supporting Information Available: Detailed biological proto-
cols, physical and spectroscopic data for compounds 5a-l, 6a-n,
10a-l, 11a-k, 12a-n, and elemental analysis results of 5a-l and
6a-n. This material is available free of charge via the Internet at
http://pubs.acs.org.
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