Design and synthesis of 2-heterocyclyl-3-arylthio-1H-indoles as potent tubulin polymerization and cell growth inhibitors with improved metabolic stability

Article abstract of DOI:10.1021/jm2012886

New arylthioindoles (ATIs) were obtained by replacing the 2-alkoxycarbonyl group with a bioisosteric 5-membered heterocycle nucleus. The new ATIs 5, 8, and 10 inhibited tubulin polymerization, reduced cell growth of a panel of human transformed cell lines

Full text of DOI:10.1021/jm2012886

Article
pubs.acs.org/jmc
Design and Synthesis of 2-Heterocyclyl-3-arylthio-1H-indoles as
Potent Tubulin Polymerization and Cell Growth Inhibitors with
Improved Metabolic Stability
Giuseppe La Regina,† Ruoli Bai,‡ Willeke Rensen,§ Antonio Coluccia,† Francesco Piscitelli,† Valerio Gatti,†
̂
̂
̂
Alessio Bolognesi,§ Antonio Lavecchia,# Ilaria Granata,∥ Amalia Porta,∥ Bruno Maresca,∥
Alessandra Soriani,∇ Maria Luisa Iannitto,∇ Marisa Mariani,○ Angela Santoni,∇,§ Andrea Brancale,⊥
Cristiano Ferlini,○ Giulio Dondio,◆ Mario Varasi,¶ Ciro Mercurio, Ernest Hamel,‡ Patrizia Lavia,§
□
Ettore Novellino,# and Romano Silvestri*,†
†Istituto PasteurFondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universita
̀
di Roma,
Piazzale Aldo Moro 5, I-00185 Roma, Italy
‡Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National
Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, United States
§CNR, National Research Council, Institute of Molecular Biology and Pathology, Sapienza Universita
̀
di Roma, Via degli Apuli 4,
I-00185 Roma, Italy
̂
∥Dipartimento di Scienze Farmaceutiche, Sezione Biomedica, Universita
Italy
̀
di Salerno, Via Ponte don Melillo, I-84084 Fisciano, Salerno,
⊥Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3NB, U.K.
#Dipartimento di Chimica Farmaceutica e Tossicologica, Universita
Napoli, Italy
̀
di Napoli Federico II, Via Domenico Montesano 49, I-80131,
∇Dipartimento di Medicina Sperimentale e Patologia, Sapienza Universita
̀
di Roma, Viale Regina Elena 324, I-00161 Roma, Italy
○Danbury Hospital Research Institute, Tumor Reproductive Biology Research Laboratory, 131 West Street, Danbury, Connecticut
06810, United States
◆NiKem Research Srl, Via Zambeletti 25, 20021 Baranzate, Milano, Italy
¶European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy
□Genextra Group, DAC SRL, Via Adamello 16, 20139 Milan, Italy
S
* Supporting Information
ABSTRACT: New arylthioindoles (ATIs) were obtained by
replacing the 2-alkoxycarbonyl group with a bioisosteric 5-
membered heterocycle nucleus. The new ATIs 5, 8, and 10
inhibited tubulin polymerization, reduced cell growth of a
panel of human transformed cell lines, and showed higher
metabolic stability than the reference ester 3. These
compounds induced mitotic arrest and apoptosis at a similar
level as combretastatin A-4 and vinblastine and triggered
caspase-3 expression in a significant fraction of cells in both
p53-proficient and p53-defective cell lines. Importantly, ATIs
5, 8, and 10 were more effective than vinorelbine, vinblastine,
and paclitaxel as growth inhibitors of the P-glycoprotein-overexpressing cell line NCI/ADR-RES. Compound 5 was shown to
have medium metabolic stability in both human and mouse liver microsomes, in contrast to the rapidly degraded reference ester
3, and a pharmacokinetic profile in the mouse characterized by a low systemic clearance and excellent oral bioavailability.
depolymerization, a process known as dynamic instability. MTs
are required for many essential cellular functions, including the
maintenance of cell shape, cell motility, intracellular transport,
INTRODUCTION
■
Microtubules (MTs) are a broadly exploited target in the search
for new effective anticancer agents.1−3 MTs are highly dynamic
cylindrical structures principally composed of α,β-tubulin
heterodimers. During their assembly, MTs continuously
undergo regulated transitions between polymerization and
Received: June 25, 2011
Published: November 1, 2011
© 2011 American Chemical Society
8394
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
and cell division. At the onset of mitosis, the interphase MTs
rapidly disassemble, and newly polymerized MTs organize into
the mitotic spindle, a symmetrical dynamic structure that drives
chromosome segregation. Thus, the mitotic apparatus is
cyclically assembled to ensure even chromosome distribution
into two genetically identical daughter cells. Interfering with
MT function, by either inhibiting tubulin polymerization or
blocking MT dynamic transitions, causes cell damage and
blocks cell division. Devising compounds that interfere with
these cellular processes is therefore an established strategy to
inhibit the proliferation of cancer cells.2−4
(B) the sulfur atom bridge, (C) the 3-arylthio group, and (D)
the substituent at position 5 of the indole (Chart 1). Previous
SAR studies at the A region addressed the elongation of the 2-
methoxy- or 2-ethoxycarbonyl group by means of C3−C5
alkoxy chains.14,15 Other ester functionalities were not explored,
as the C3−C5 alkoxy chains led to decreases of both
antitubulin and antiproliferative activities.15
In studies to be presented here, we found that ester
derivative 3 was extensively degraded by mouse and human
liver microsomes. Therefore, we decided to replace the
ethoxycarbonyl group of 3 (A region) with a potentially
more stable five-membered heterocyclic nucleus, the bioisos-
teric pyrrole, furan, and thiophene moieties.18 Preliminary
modeling studies showed that, with respect to the small/
medium linear chain,15 the five-membered heterocycle at
position 2 of the indole nucleus could form new hydrophobic
interactions with Lys254 and Leu248 of the colchicine site of
tubulin (Supporting Information).
MT assembly can be inhibited by using colchicine (1),5,6
combretastatin A-4 (CSA4, 2)7 (Chart 1), and the Cathar-
Chart 1. Structures of ATI Derivatives 3−12 and Reference
a
Compounds 1 and 2
Here, we report that this strategy was successful. The
analogues with a pyrrole (4, 5), furan (6, 7), or thiophene (8−
12) moiety had enhanced activity against the target tubulin, and
moreover, compound 5, in particular, displayed much better
stability than 3 in the microsomal assays and good
pharmacokinetic properties.
CHEMISTRY
■
Microwave (MW) reaction of an appropriate 2-heterocyclyl-
1H-indole 13,19 14−17, or 19 with bis(3,4,5-
trimethoxyphenyl)disulfide13 in the presence of sodium hydride
in anhydrous DMF at 110 °C (150 W) for 2 min furnished 4−8
or 12 (Scheme 1).
a
A−D: ATIs’ binding regions. R1 = 1H-pyrrol-2-yl, 1H-pyrrol-3-yl,
furan-2-yl, furan-3-yl, thiophen-2-yl, thiophen-3-yl; R2 = H, OMe; X =
S, CO, CH2.
a
Scheme 1. Synthesis of Compounds 4−12
anthus alkaloids vincristine and vinblastine (VBL), all of which
prevent tubulin polymerization. This results in MT destabiliza-
tion, arrest of mitotic progression, and subsequent cell death.
Another class of antimitotic drugs, including taxoids and
epothilones, target a lumenal site on the β-subunit8,9 and enter
the lumen through a binding site10 located at a pore on the MT
surface formed by different tubulin heterodimers. In that group,
paclitaxel (PTX) stimulates MT polymerization and stabiliza-
tion at high concentrations, whereas at lower concentrations it
inhibits MT dynamics with little effect on the proportion of
tubulin in polymer.11 Thus, either inhibition or enhancement of
tubulin assembly prevents proper functioning of the mitotic
apparatus and ultimately blocks cell division.
MT-targeting drugs with different mechanisms of action have
an empirical therapeutic efficacy in a variety of tumor types.
Nevertheless, a number of unsolved problems still remain in
their clinical use: (i) drug resistance appears often, with a
poorly understood molecular basis, which makes it a difficult to
predict phenomenon; (ii) secondary toxicity on nontrans-
formed cells is often observed in clinical practice, partly
independent of the antitubulin mechanism of action of any
specific drug.12 There is therefore an urgent need to design and
synthesize novel tubulin inhibitors of improved efficacy.13
Arylthioindole (ATI) antimitotic agents are potent inhibitors
of tubulin polymerization and cancer cell growth. ATIs inhibit
[3H]colchicine binding in the β-tubulin site close to its interface
with α-tubulin within the α,β-dimer.14 Structure−activity
relationship (SAR) studies of ATIs14−17 have been focused
on the following: (A) the substituent at position 2 of the indole,
a
4−12; see Table 1. 13:24 R1 = 1H-pyrrol-2-yl, R2 = H. 14: R1 = 1-
(benzenesulfonyl)-1H-pyrrol-3-yl, R2 = H. 15: R1 = furan-2-yl, R2 = H.
16: R1 = furan-3-yl, R2 = H. 17: R1 = thiophen-2-yl, R2 = H. 18: R1 =
thiophen-2-yl, R2 = OMe. 19: R1 = thiophen-3-yl, R2 = H. Reagents
and reaction conditions: (a) (for 4−8 and 12) (i) NaH, anhydrous
DMF, 25 °C, 10 min; (ii) bis(3,4,5-trimethoxyphenyl)disulfide, closed
vessel, 150 W, 110 °C, 2 min, yield 10−83%; (b) (for 9) (i) NaH,
anhydrous DMF, 0 °C, Ar stream, 15 min; (ii) bis(3,4,5-
trimethoxyphenyl)disulfide, 110 °C, Ar stream, overnight, yield 70%;
(c) (for 10) 3,4,5-trimethoxybenzoyl chloride, anhydrous AlCl3, 1,2-
dichloroethane, closed vessel, 150 W, 110 °C, 2 min, yield 34%; (d)
(for 11) NaBH4 (10 equiv), ethanol, reflux, 2.5 h, yield 21%.
Compound 10 was prepared from 17 and 3,4,5-trimethox-
ybenzoyl chloride in the presence of anhydrous aluminum
chloride in 1,2-dichloroethane at 110 °C (150 W) for 2 min.
Sodium borohydride (10 equiv) reduction of 10 in boiling
ethanol provided 11. Derivative 9 was obtained by heating the
8395
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
corresponding indole 18 with the disulfide13 in the presence of
sodium hydride in anhydrous DMF.
The acid chloride of 20 was treated with 1-(phenylsulfonyl)-
1H-pyrrole in the presence of anhydrous aluminum chloride to
give the intermediate 21, which underwent intramolecular
cyclization to 14 with iron powder in glacial acetic acid at 60 °C
(Scheme 2a). Treatment of 2-acetylfuran with phenylhydrazine
acid in the presence of Pd(II) acetate and potassium carbonate
in a closed vessel at 110 °C (200 W) for 15 min gave 16
(Scheme 2c).
2-Acetyl- (25) and 3-acetylthiophene (27) were brominated
to 26 and 28 and transformed into 17 or 19, respectively, by
reaction with aniline in the presence of a catalytic amount of
DMF in a closed vessel at 100 °C (150 W) for 1 min (Scheme
2d and e). Reaction of (5-methoxy-2-nitrobenzyl)-
trimethylsilane21 (29) with thiophen-2-carboxaldehyde in the
presence of tetrabutylammonium fluoride (TBAF) provided
alcohol 30, which was oxidized to 31 with pyridinium
chlorochromate. Tin(II) chloride reduction of 31 and
subsequent intramolecular cyclization of the amino intermedi-
ate furnished 18 (Scheme 2f).
a
Scheme 2. Synthesis of Compounds 14-19
RESULTS AND DISCUSSION
■
Inhibition of Tubulin Polymerization. In a first set of
experiments, we assessed the ability of ATIs 4−12 to inhibit
tubulin polymerization in vitro (Table 1). The new ATIs
inhibited tubulin polymerization with IC50 values ranging from
0.74 to 1.9 μM, as compared with 1.0 μM for CSA4 (2), 3.2
μM for colchicine (1), and 2.9 μM for compound 3.14 As
tubulin polymerization inhibitors, 6−10 and 12 were equal to
or more potent than 2, with the most potent inhibitor being 8,
which had an IC50 of 0.74 μM. Replacement of the sulfur
bridging atom of 8 with a carbonyl functionality led to ketone
10, which inhibited tubulin polymerization with an IC50 of 1.0
μM.
The new ATIs were also examined for potential inhibition of
the binding of [3H]colchicine to tubulin (Table 1). All except
11 were strong inhibitors of the binding reaction (75−92%
inhibition, although not quite as potent as CSA4). In this assay,
the strongest inhibition was observed with compounds 4 and 7.
Cell Growth Inhibition. All the new ATIs, except
compound 11, inhibited the growth of human MCF-7
nonmetastatic breast cancer epithelial cells with IC50 values
ranging from 18 to 60 nM, with the most active compounds
being 4 and 5 (Table 1). The antiproliferative effect of
compounds 5 and 8−11 was evaluated in four additional cancer
cell lines in comparison with 2 and doxorubicin (DOX), a
DNA-targeting drug often employed in association with
antimitotic agents (Table 2). Compounds 8 and 10 were
generally 1 order of magnitude more potent as growth
inhibitors of HeLa (cervix), PC3 (prostate), HT-29 (colon),
A549 (nonsmall cell lung), and 231-MDA (metastatic breast)
carcinoma-derived cell lines as compared with DOX. The effect
of 9 went from 2- to 3-fold more potent than DOX to
equimolar, depending on the cell line.
Compound 5 was highly effective in the HT-29 and A549
cell lines and had efficacy comparable with that of 9 in the
HeLa and PC3 cell lines. As growth inhibitors of HeLa and
PC3 cells, compounds 8 and 10 were more potent than 2, while
5 and 9 were generally less potent. As compared with 2,
compounds 5 and 8-11 were superior as inhibitors of the
growth of HT-29 and A549 cell lines. Compound 11 was
generally comparable with DOX, but it was less potent than 2
as inhibitor of HeLa and PC3 cells growth. These data
underscore the effectiveness of ATIs 5 and 8−10 in
transformed cell types. Such results also highlighted a
differential sensitivity to MT-targeting drugs displayed by cell
lines with different genetic backgrounds, consistent with results
obtained with other drugs that target the mitotic apparatus.22,23
a
Reagents and conditions: (a) (i) oxalylchloride, catalytic anhydrous
DMF, 1,2-dichloroethane, 0 °C, Ar stream, 30 min; (ii) 1-
(phenylsulfonyl)-1H-pyrrole, AlCl3, 0 °C, Ar stream, 15 min, yield
10%; (b) Fe, AcOH, 60 °C, overnight, yield 10%; (c) phenylhydrazine
hydrochloride, anhydrous CH3COONa, ethanol, open vessel, 250 W,
PowerMAX, 80 °C, 5 min, yield 94%; (d) PPA, 110 °C, 1 h, yield 55%;
(e) 3-furan boronic acid pinacol ester, Pd(II) acetate, potassium
carbonate, 1-methyl-2-pyrrolidinone/water, closed vessel, 200 W, 110
°C, 15 min, yield 95%; (f) bromine, dichloromethane, 25 °C, 1 h, yield
57−80%; (g) (i) aniline, 25 °C, 3 h; (ii) catalytic anhydrous DMF,
closed vessel, 150 W, 100 °C, 1 min, yield 16−40%; (h) thiophen-2-
carboxaldehyde, TBAF, anhydrous THF, 25 °C, Ar stream, 15 min,
yield 61%; (i) pyridinium chlorochromate, anhydrous CH2Cl2, 25 °C,
1.5 h, yield 56%; (j) tin(II) chloride dihydrate, AcOEt, reflux, 3 h, yield
21%.
hydrochloride in the presence of anhydrous sodium acetate in
an open vessel at 80 °C (250 W) for 5 min furnished hydrazone
23, which was converted to 15 by heating at 110 °C with
polyphosphoric acid (PPA) for 1 h (Scheme 2b). Reaction of 2-
iodo-1H-indole20 (24) with the pinacol ester of furan-3-boronic
8396
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
Table 1. Inhibition of Tubulin Polymerization, Growth of MCF-7 Human Breast Carcinoma Cells, and Colchicine Binding by
Compounds 1−12
a
b
Inhibition of tubulin polymerization. Tubulin was 10 μM during polymerization. Inhibition of growth of MCF-7 human breast carcinoma cells.
c
d
Inhibition of [3H]colchicine binding: tubulin was at 1 μM, both [3H]colchicine and inhibitor were at 5 μM. Reference 11.
Table 2. Inhibition of Growth of HeLa, PC3, HT29, and
A549 Cell Lines by Compounds 5 and 8−11
Table 3. Growth Inhibition in A2780wt Cells, Its Cisplatin
Resistant Counterpart A2780-CIS, and OVCAR-3 by
Compounds 5 and 10
a b
,
IC50 ± SD (μM)
IC50 ± SD (nM)
compd
HeLa
PC3
HT-29
A549
a
b
compd
A2780wt
A2780-CIS
OVCAR-3
RI-1
RI-2
5
0.4 ± 0.02
0.09 ± 0.002
0.4 ± 0.02
0.07 ± 0.004
1 ± 0.03
0.5 ± 0.1
0.2 ± 0.05
0.8 ± 0.08
0.1 ± 0.08
2 ± 0.1
0.1 ± 0.03
0.15 ± 0.03
0.4 ± 0.02
0.08 ± 0.01
1 ± 0.05
0.08 ± 0.02
0.09 ± 0.02
0.3 ± 0.09
0.08 ± 0.01
2 ± 0.08
8
5
21.5 ± 1.2
30.5 ± 0.7
485 ± 60
6.3 ± 1.8
29.3 ± 3.2
8980 ± 565
81 ± 21
117 ± 18
1816 ± 254
0.29
0.96
18.5
3.8
3.8
3.7
9
10
10
11
2
cisplatin
a
RI-1 (Resistant Index-1) was calculated by dividing the IC50 obtained
b
0.2 ± 0.005
1.5 ± 0.03
0.4 ± 0.02
1.7 ± 0.02
>10
>10
in A2780-CIS by the IC50 obtained in A2780wt. RI-2 (Resistant
Index-2) was calculated by dividing the IC50 obtained in OVCAR-3 by
the IC50 obtained in A2780wt. A value >1 or <1 means either
increased cisplatin-resistance or sensitivity, respectively.
c
DOX
1.0 ± 0.04
1.0 ± 0.08
a
Growth inhibition of the indicated cell lines (MTT method);
incubation time was 48 h. Plots of 231-MDA and A549 cell growth
b
inhibition by compounds 5 and 8−10 are shown in Figures 1S−4S of
c
other hand, such an increase was not detectable in the
endogenous resistance model, as the resistance values (RI-2)
for the two ATIs were essentially identical to that obtained with
cisplatin in OVCAR-3.
Pgp Overexpressing MDR Cell Lines. Compounds 5, 8,
and 10 were compared with vinorelbine (VRB), vinblastine
(VBL), PTX, and 2 in the OVCAR-8 and NCI/ADR-RES cell
lines (Table 4). Derived from OVCAR-8, NCI/ADR-RES is a
DOX-resistant cell line that overexpresses P-glycoprotein
the Supporting Information. Doxorubicin as reference compound.
We analyzed growth inhibition in an ovarian cancer model
with high drug sensitivity (A2780wt), its cisplatin-resistant
counterpart (A2780-CIS), and a cell line derived from a
cisplatin-resistant patient (OVCAR-3) (Table 3). For the
acquired cisplatin-resistance (RI-1) model, compounds 5 and
10 yielded even lower IC50 values than in the parental line, in
contrast to the almost 20-fold resistance to cisplatin. On the
8397
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
and 8−11 were analyzed for their effects on the cell cycle in
parallel with VBL and 2 (Figure 1). Twenty-four hours after
plating, HeLa cells were exposed to each compound for 24 h,
and the cell cycle profile was subsequently analyzed by flow
cytometry. We found that, for 5, 8, and 10 at 100 nM, live cell
imaging (Figure 1, column A) indicates that cultures exposed to
these ATIs progressed in the cell cycle up to the point where
they would normally assemble the mitotic apparatus; at this
point, the ATIs inhibited this assembly and prevented further
progression into mitosis, inducing 70−90% of all the cells to
accumulate with a G2/M DNA content (Figure 1, panels B−
C), similar to VBL and 2 (80−90% G2/M arrest).
Table 4. Growth Inhibition of OVCAR-8 and NCI/ADR-
RES Cells Lines by Compounds 5, 8, and 10 and Reference
Compounds VRB, VBL, PTX, and 2
a
IC50 ± SD (nM)
b
c
compd
OVCAR-8
NCI/ADR-RES
5
70 ± 30
45 ± 20
20 ± 10
300 ± 0
15 ± 7
25 ± 7
25 ± 7
15 ± 7
5000 ± 1000
200 ± 0
8
10
d
VRB
e
VBL
PTX
2 ± 0.7
1.3 ± 0.6
1500 ± 700
1.3 ± 0.6
2
The cell cycle effects of ATIs 8 and 10 were further examined
in the A549 and HT29 cell lines. Both 8 and 10 induced a
gradual accumulation of cells in the G2/M phase of the cell
cycle in a dose-dependent manner, and this could be detected
as early as 7 h after initiation of treatment. In these cells we
evaluated both the cell cycle profile and the accumulation of
cyclin B1, following treatment with the compounds (Figure 2).
Mitotic Index. Both flow cytometry and the Western blot
assay for cyclin B1 provide information about the entire cell
population. Yet, these experiments do not provide definitive
proof that the cells are actually arrested in mitosis. True arrest
in mitosis would be a desirable outcome of tubulin inhibitory
treatment from a therapeutic perspective, because (1), by
definition, dividing cells would be selectively targeted while
resting, interphase cells would be spared and (2) sustained
mitotic arrest can trigger a specific cell death pathway with
molecular features, such as the independence from p53,
common to many tumors.26 The mitotic cell death pathway
can therefore operate effectively in tumor cells, including those
harboring p53 mutations, differently from the classical
apoptotic response induced by DNA damage that requires
functional p53. To ascertain whether ATIs with cell cycle
inhibitory activity induce true mitotic arrest, we processed
treated cells for immunofluorescence by staining with an
antibody directed against α-tubulin and with the DNA dye 4′,6-
diamidino-2-phenylindole (DAPI). Treated and control cells
were then scored for their mitotic index (MI), calculated as
mitotic cells/(mitotic + interphase cells). In control cultures,
about 10% of cells were in the various stages of mitosis, with
well-formed spindles interacting with condensed chromosomes
(Figure 3A). In cultures exposed to ATI 5, 8, or 10, over 60%
of the cells were arrested in a prometaphase-like state, with
condensed chromosomes and sparse foci of tubulin that failed
to elongate to form the spindle microtubules (Figure 3B).
Thus, the accumulation of G2/M cells detected by flow
cytometry reflected a true mitotic arrest. The MI in cell
populations treated with 5, 8, and 10 was significantly higher
compared with treatment with the weak tubulin inhibitor 11.
The MIs observed with 5 and 10 were in the same range as
with 2 and VBL, although a higher concentration of the ATIs
was required (Figure 3C). Compound 9 was less effective in
inducing mitotic arrest and reproducibly yielded a MI of about
35%.
a
Cells were grown in RPMI 1640 medium with 5% FBS, 5% CO2
b
atmosphere at 37 °C, for 96 h. OVCAR-8: ovarian tumor cell line 8.
c
NCI/ADR-RES: DOX-resistant cell line derived from OVCAR-8.
d
Vinorelbine, tubulin assembly inhibition = 3.1 ± 0.2 μM.
e
Vinblastine, tubulin assembly inhibition = 1.1 ± 0.2 μM.
(Pgp), resulting in the type 1 multidrug-resistance phenotype.
Compounds 5, 8, and 10 were uniformly more active in the
NCI/ADR-RES line than in the parental OVCAR-8 line. In
contrast, VRB and VBL showed the typical multidrug resistance
differential, as did PTX.
CSA4 yielded the same IC50 in both cell lines and clearly was
not a Pgp substrate. Most importantly, as NCI/ADR-RES cell
growth inhibitors, ATIs 5, 8, and 10 were all superior to VRB,
VBL, and PTX. These compounds were from 200- (5 and 8) to
333-fold (10) more potent than VRB, from 13- (5 and 8) to 8-
fold (10) more potent than VBL, and from 100- (5 and 8) to
60-fold (10) more potent than PTX.
Nontransformed Cell Lines. A recurrent problem with
MT-targeting drugs is their widespread toxicity on normal cells
when used in human patients.2,24,25 We therefore used
nontransformed cells to evaluate potential differences between
compounds 5 and 11, as representatives of one of the more
potent ATIs and the least potent in the current series (Table
Table 5. Growth Inhibition of MCF-7, HOSMAC, A10,
PtK2, and HUVEC Cell Lines by Compounds 2, 5, and 11
and PTX
a
IC50 ± SD (nM)
b
c
d
e
compd
MCF-7
HAOSMC
A10
PtK2
HUVEC
5
20 ± 0
200 ± 0
13 ± 3
33 ± 10
250 ± 90
5 ± 3
40 ± 30
150 ± 90
1 ± 1
60 ± 0
300 ± 0
3 ± 1
30 ± 0
180 ± 40
1 ± 1
11
2
f
PTX
3 ± 0.5
6 ± 6
38 ± 10
21 ± 8
7 ± 4
a
SD of 0 indicates the same value was obtained in all experiments.
HAOSMC: human aortic smooth muscle cells (ATCC #CLR-1999).
b
c
A10, rat embryonic aortic smooth muscle cells (ATCC #CRL-1476).
d
PtK2: Potorous tridactylis kidney epithelial cells (ATCC #CLL-56).
e
HUVEC: human umbilical vein endothelial cells (ATCC #CRL-
f
2873). Data from ref 16.
5). Both compounds 5 and 11 had somewhat greater
antiproliferative effects in MCF-7 breast cancer cells as
compared with several nontransformed cell lines, with the
difference being greater for 5 than 11. Both compounds
showed less differential activity, however, than did PTX, and 2
was the least selective of the compounds evaluated.
Caspase-3 Expression. The data at this point indicated
that most ATIs that effectively inhibited tubulin polymerization
in vitro arrested mitotic progression in cultured cells, thereby
causing decreased cell proliferation in human transformed cell
lines. To investigate more closely the link between mitotic
arrest and reduced cell growth, we analyzed HeLa cell
populations exposed to 5 and 8−11 for induction of cell
death under the same conditions used in the mitotic arrest
Cell Cycle Analysis. To further characterize the cell
growth inhibitory properties of the new ATIs, compounds 5
8398
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
Figure 1. Effect of ATIs on HeLa cell cycle progression. (A) Bright-field panels from HeLa cell cultures exposed for 24 h to solvent (DMSO) or to
the indicated compounds (VBL and 2 are shown at 20 nM; all other compounds at 100 nM). Rounded, highly refractive cells blocked in mitosis are
clearly distinguishable from flattened interphase cells (10× objective). (B) Representative flow cytometry profiles of cultures exposed to compounds
at the same concentrations as in part A. Cells stained with propidium iodide (PI) were separated according to their DNA content, calculated from
the emitted PI fluorescence (x axis); the y axis indicates the number of cells. The 2C peak (diploid DNA content) identifies G1 cells; the 4C peak
(tetraploid DNA content) identifies G2/M cells. Cells with intermediate DNA contents are in the S phase. C. Bars represent the percent of G2/M
cells (4C genomic content) (mean values ± SD were from two to six independent experiments).
experiments. Control cultures were compared with cultures
treated with ATIs 5, 8, and 10 for their reactivity to annexin V,
which binds to phosphatidylserine residues that are translocated
from the inner to the outer cell membrane in early apoptosis.
Annexin V-reactive cells were quantitated by flow cytometry
(Figure 4A). The results showed that 5, 8, and 10 induced
apoptosis as efficiently as 2 and VBL at the concentrations
examined. To extend these results, we analyzed the induction of
caspase-3 expression at the single cell level. Caspase-3 regulates
the induction of apoptosis in mitotic cells, and it is required for
the apoptotic response to MT-targeting drugs.1,27 Immuno-
fluorescence showed that 5, 8, and 10, but not 11, induced
caspase-3 activity in a significant fraction of cells (an apoptotic
cell treated with 10 is shown in Figure 4B), further
demonstrating the strong link between the ability of ATIs to
arrest mitotic progression and to induce apoptosis.
Metabolic Stability. An initial study with compound 3
confirmed our fears that it would be metabolically unstable,
arguing against its potential as a therapeutic agent. With both
mouse and human microsomal preparations, 3 was totally
degraded in 30 min (Table 6). Having prepared 5, 8, and 10
and the other five-member heterocycle ATIs with the idea that
they should overcome this metabolic instability, we examined
these compounds in a standard microsomal stability assay
(Table 6), in comparison with the control compounds 7-
ethoxycoumarin and propanolol. This assay, using both human
and mouse liver microsomes, estimates compound stability to
phase I oxidative metabolism. Compounds 5 and 10 showed
8399
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
Figure 3. Induction of mitotic arrest by ATIs. (A) HeLa control
cultures exposed to DMSO solvent (0.1%, 24 h). MTs were visualized
by α-tubulin staining (green) and chromosomes by DNA staining
(blue). Most cells were in the interphase. Cells in mitosis (PM,
prometaphase; M, metaphase) are indicated. (B) A typical field from a
HeLa culture exposed to ATI 10 (100 nM, 24 h). Note that most cells
were arrested in early mitotic stages, with condensed chromosomes
and a failure to assemble proper MTs. Disorganized tubulin foci are
seen throughout the mitotic cells. (C) MIs in HeLa cell cultures that
were treated with DMSO solvent, with known anti-MT compounds, or
with ATIs 5 and 8−11 (mean values ± SD were calculated from two
to four independent experiments).
Figure 2. Dose-dependent cell cycle effects of ATI 10 in A549 and
HT29 cell cultures. (A) Typical cell cycle profiles after flow cytometric
analysis of A549 cell populations treated with DMSO only or with ATI
10 (100 nM, 7 h). (B) The histograms represent the percent of cells
with G1, S, and G2/M DNA content, based on flow cytometric
analyses of A549 cells exposed to increasing micromolar concen-
trations of ATIs 8 and 10 (7 h). (C) Cyclin B1 accumulation in A549
and HT29 cell lines exposed to the indicated micromolar
concentrations of 10 (7 h), with actin (Act) as a loading control
(Santa Cruz Biotechnology).
iv and os Pharmacokinetics. Finally, the intravenous
pharmacokinetic and oral bioavailability of 5 was investigated in
the mouse. Pharmacokinetic studies were carried out in male
CD-1 mice. The compound was administered either in single
intravenous (iv) injections of 5 mg/kg or in an oral (os) dose of
15 mg/kg. The main pharmacokinetic parameters obtained are
summarized in Table 7. Compound 5 showed a systemic
plasma clearance slightly lower than the hepatic blood flow of
86 mL/min·kg in mice, an estimated elimination half-life of 40
min, and a calculated steady state volume of distribution
suggestive of good tissue distribution. Moreover, the compound
was quickly absorbed after oral administration and showed a
very high oral bioavailability. Profiles of distribution in plasma
of 5 after iv and os administration and a comparison of the
kinetic profiles are shown in Figures 5S and 6S, respectively, in
the Supporting Information.
medium and low metabolic stability (see Table 6 legend for
definitions of relative stabilities), respectively, with both the
human and mouse liver microsomes, while 8 exhibited medium
stability with the human microsomes and low stability with the
mouse microsomes. The metabolic stability of 5, 8, and 10 may
be dependent on the nature of the 2-heterocyclic nucleus rather
than the structure of the bridging group. Such an effect may be
explained by the steric hindrance exerted by the two aromatic
moieties attached to the bridging group.
Aqueous Solubility. The aqueous solubility of 5 was 4
times greater than that of compound 3, as a result of the
substitution of the pyrrole nucleus for the ester moiety. The
aqueous solubility of 5 at pH 7.4 was 20 μM, while under the
same conditions the solubility of 3 was 5 μM. This
improvement in the aqueous solubility of 5 may be the
explanation for the greater activity of 5 relative to 3 as an
inhibitor of tubulin polymerization and MCF-7 cell growth,
although the molecular modeling studies presented above
suggest that 5 should bind more tightly than 3 to tubulin.
CONCLUSION
■
We designed new ATIs by replacing the 2-methoxy-/
ethoxycarbonyl group (A region) with a bioisosteric 5-
membered heterocycle nucleus. The newly synthesized ATIs
showed effective tubulin inhibitory properties in vitro and
potential therapeutic value in cancer treatment. The ATIs
described here have significantly improved efficacy compared
with the previously synthesized 3 used as a reference and
compared well with classical MT inhibitors. In particular, the
new ATIs 5, 8, and 10 showed a valuable biological profile: (i)
they were potent tubulin polymerization inhibitors in the low/
8400
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
Table 7. Pharmacokinetic Properties of 5 in Mice
parameter
value
a
Cmax (iv) (ng/mL)
35.67
174346
40
b
AUC0−∞ (iv) (ng·min/mL)
t1/2 (min)
c
d
Cl (mL/min·kg)
28.6
e
Vss (L/kg)
1.59
a
Cmax (os) (ng/mL)
1340
120
f
t
max (os) (min)
b
AUC0−∞ (os) (ng·min/mL)
(%)
583315
110
g
F
a
b
Maximum concentration. Area under the curve calculated up to the
c
d
e
0−∞ time point. Half-time. Clearance. Mean distribution volume.
f
g
max-time. Oral bioavailability.
transformed cell lines via arrest of mitotic progression and
induction of cell death; (v) the induction of mitotic arrest in
cell populations treated with these ATIs was in the same range
as was observed with CSA4 and VBL; (vi) they induced
apoptosis in the same range or above the level induced by VBL
and CSA4 and triggered caspase-3 activation; (vii) the sulfur
bridging group of ATIs showed satisfactory metabolic stability.
We would like to highlight their effectiveness in the HeLa and
231-MDA cell lines, both of which lack functional p53: this
indicates that the cell death pathway triggered by ATIs is p53-
independent. This is of relevance from a therapeutic
perspective, given that about 50% of all human tumors are
defective for p53 function and cannot respond to DNA-
targeting drugs, which induce p53-dependent apoptosis. On the
whole, the present data strongly support the therapeutic
potential and further development of the new ATIs described
here. Finally, the metabolic stability of compound 5, obtained
in both mouse and human microsomes, along with the excellent
mouse pharmacokinetic profile, warrant further in vivo efficacy
studies.
Figure 4. Induction of apoptosis by ATIs. (A) Typical flow cytometry
studies of HeLa cell populations incubated with annexin V after
exposure to DMSO only (control) or to 10 (100 nM). The histograms
represent the percent apoptotic cells, as determined by annexin V-
reactivity, after exposure to the indicated compounds for 24 h (means
± SD were calculated from two to three experiments). (B) Single-cell
level immunofluorescence analysis revealed induction of active
caspase-3 (recognized by a specific antibody). The panels depict
typical fields from HeLa cultures exposed to only DMSO (note the
two cells progressing through mitosis) or to 10 (a mitotic cell
expressing active caspase-3 is indicate by the arrow among a group of
cells arrested in mitosis).
EXPERIMENTAL SECTION
■
Chemistry. MW-assisted reactions were performed on a Discover
S-Class (CEM) single mode reactor, controlling instrument
parameters with PC-running Synergy 1.4 software. Temperature,
irradiation power, maximum pressure (Pmax), PowerMAX (in situ
cooling during the MW irradiation), ramp and hold times, and open
and closed vessel modes were set as indicated. Reactions in an open
vessel were carried out in 100 mL round-bottom flasks equipped with
a Dimroth reflux condenser and a cylindrical stirring bar (length 20
mm, diameter 6 mm). Reactions in a closed vessel were performed in
capped MW-dedicated vials (10 mL) with a cylindrical stirring bar
(length 8 mm, diameter 3 mm). The temperature of the reaction was
monitored with a built-in infrared (IR) sensor. After completion of the
reaction, the mixture was cooled to 25 °C via air-jet cooling. Melting
points (mp's) were determined on a SMP1 apparatus (Stuart
Scientific) and are uncorrected. IR spectra were run on a
SpectrumOne FT-ATR spectrophotometer (Perkin-Elmer). Band
position and absorption ranges are given in inverse centimeters.
Proton nuclear magnetic resonance (1H NMR) spectra were recorded
on a 400 MHz FT spectrometer (Bruker) in the indicated solvent.
Chemical shifts are expressed in δ units (ppm) from tetramethylsilane.
Mass spectra were recorded on a Bruker MicroTOF LC column.
Column chromatography was performed on columns packed with
alumina from Merck (70−230 mesh) or silica gel from Macherey-
Nagel (70−230 mesh). Aluminum oxide thin layer chromatography
(TLC) cards from Fluka (aluminum oxide precoated aluminum cards
with fluorescent indicator visualizable at 254 nm) and silica gel TLC
cards from Macherey-Nagel (silica gel precoated aluminum cards with
fluorescent indicator visualizable at 254 nm) were used for TLC.
Table 6. Metabolic Stability of 5, 8, and 10 with Human and
a
Mouse Liver Microsomes
b
% remaining at 30 min
compd
human liver microsomes mouse liver microsomes
5
34.8 ± 1.4
22.0 ± 1.0
4.6 ± 0.2
0.37 ± 0.01
6.6 ± 0.2
23.1 ± 0.6
2.2 ± 0.3
0.60 ± 0.01
0.5 ± 0.03
0.07 ± 0.02
20.6 ± 0.5
8
10
3
c
7-ethoxycoumarin
c
propanolol
54.1 ± 0.4
a
Metabolic stability: >95, high; 50−95, good; 10−50, medium; <10,
b
c
low. Results are expressed as mean ± SD, n = 2. The standard
compounds 7-ethoxycoumarin and propanolol showed metabolic
stability in agreement with the literature and internal validation data.28
submicromolar range of concentration; (ii) such compounds
were uniformly more active in the Pgp-overexpressing NCI/
ADR-RES cell line than in the parental OVCAR-8 line and were
superior to VRB, VBL, and PTX; (iii) compounds 5 and 10
showed selective activity against cells with acquired cisplatin
resistance; (iv) they reduced cell growth in a panel of human
8401
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
Developed plates were visualized with a Spectroline ENF 260C/FE
UV apparatus. Organic solutions were dried over anhydrous sodium
5-Methoxy-2-(thiophen-2-yl)-3-[(3′,4′,5′-trimethoxyphenyl)-
thio]-1H-indole (9). 18 (0.19 g, 0.00083 mol) was added at 0 °C to
a suspension of sodium hydride (60% in mineral oil, 0.05 g, 0.0012
mol) in anhydrous DMF (2 mL). After 15 min, bis(3,4,5-
trimethoxyphenyl)disulfide14 (0.36 g, 0.00091 mol) was added, and
the reaction was heated at 110 °C overnight under an Ar stream. After
cooling, the mixture was carefully diluted with water and extracted
with ethyl acetate. The organic layer was washed with brine and dried.
Removal of the solvent gave a residue that was purified by column
chromatography (silica gel, ethyl acetate/n-hexane 1:2 as eluent) to
furnish 9 as a white solid (0.25 g, 70%), mp 55−58 °C (from ethanol).
1H NMR (CDCl3): δ 3.67 (s, 6H), 3.78 (s, 3H), 3.84 (s, 3H), 6.39 (s,
2H), 6.92 (dd, J = 2.5 and 8.7 Hz, 1H), 7.11−7.13 (m, 2H), 7.31 (dd, J
= 0.5 and 8.7 Hz, 1H), 7.39 (dd, J = 1.1 and 5.1 Hz, 1H), 7.52 (dd, J =
1.1 and 3.7 Hz, 1H), 8.54 ppm (br s, 1H, disappeared on treatment
with D2O). MS: ES+ = 428 (MH+). IR: ν 3301 cm−1. Anal.
(C22H21NO4S2 (427.54)) C, H, N, S.
sulfate. Evaporation of the solvents was carried out on a Buchi
̈
Rotavapor R-210 equipped with a Buchi V-850 vacuum controller and
̈
̈
Buchi V-700 and V-710 vacuum pumps. Elemental analyses of the new
compounds were found within 0.4% of the theoretical values. The
purity of tested compounds was >95%. Compounds 20, 22, 25, and 27
were purchased from Sigma-Aldrich.
General Procedure for the Preparation of Compounds 4−8
and 12. Example. 2-(1H-Pyrrol-2-yl)-3-[(3′,4′,5′-
trimethoxyphenyl)thio]-1H-indole (4). 2-(1H-Pyrrol-2-yl)-1H-in-
dole19 (13) (0.25 g, 0.0014 mol) was carefully added to a suspension
of sodium hydride (60% in mineral oil, 0.13 g, 0.0031 mol) in
anhydrous DMF (2 mL) while stirring. After 10 min, bis(3,4,5-
trimethoxyphenyl)disulfide13 (0.62 g, 0.0015 mol) was added, and the
reaction mixture was placed into the MW cavity (closed vessel mode,
Pmax = 250 PSI). MW irradiation of 150 W was used, the temperature
being ramped from 25 to 110 °C while stirring. Once 110 °C was
reached, taking about 1 min, the reaction mixture was held there for 2
min. The mixture was diluted with water and extracted with ethyl
acetate. The organic layer was washed with brine and dried. Removal
of the solvent gave a residue that was purified by column
chromatography (alumina, chloroform as eluent) to furnish 4 (0.1 g,
19%), mp 215−218 °C (from ethanol). 1H NMR (DMSO-d6): δ 3.52
(s, 6H), 3.55 (s, 3H), 6.18 (t, J = 2.9 Hz, 1H), 6,34 (s, 2H), 6.81 (d, J
= 2.4 Hz, 1H), 6.96 (br s, 1H), 7.04−7.08 (m, 1H), 7.12−7.16 (m,
1H), 7.42 (d, J = 8.1 Hz, 2H), 11.11 (br s, 1H, disappeared on
treatment with D2O), 11.64 ppm (br s, 1H, disappeared on treatment
with D2O). MS: ES+ = 381 (MH+). IR: ν 3329, 3422 cm−1. Anal.
(C21H20N2O3S (380.46)) C, H, N, S.
[ 2 - ( T h i o p h e n - 2 - y l ) - 1 H - i n d o l - 3 - y l ] - ( 3 ′ , 4 ′ , 5 ′ -
trimethoxyphenyl)methanone (10). A mixture of 17 (0.25 g,
0.00125 mol), 3,4,5-trimethoxybenzoyl chloride (0.29 g, 0.00125 mol),
and anhydrous aluminum chloride (0.17 g, 0.00125 mol) in anhydrous
1,2-dichloroethane (2 mL) was placed into the MW cavity (closed
vessel mode, Pmax = 250 PSI). MW irradiation of 150 W was used, the
temperature being ramped from 25 to 110 °C with stirring. Once 110
°C was reached, taking about 1 min, the reaction mixture was held
there for 2 min. The reaction mixture was quenched on 1 N HCl/
crushed ice and extracted with chloroform. The organic layer was
washed with brine and dried. Removal of the solvent gave a residue
that was purified by column chromatography (silica gel, ethyl acetate/
n-hexane 3:7 as eluent) to furnish 10 as a yellow solid (0.17 g, 34%),
2-(1H-Pyrrol-3-yl)-3-[(3′,4′,5′-trimethoxyphenyl)thio]-1H-in-
1
mp 155−159 °C (from ethanol). H NMR (DMSO-d6): δ 3.75 (s,
dole (5). Synthesized as 4, starting from 14, yield 10% as a brown
6H), 3.88 (s, 3H), 6.96−6.98 (m, 1H), 7.08 (s, 2H), 7.20−7.24 (m,
2H), 7.30−7.34 (m, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 7.8 Hz,
1H), 8.73 ppm (br s, 1H, disappeared on treatment with D2O). MS:
ES+ = 394 (MH+). IR: ν 1570, 3214 cm−1. Anal. (C22H19NO4S
(393.46)) C, H, N, S.
1
solid, mp 200−204 °C (from ethanol). H NMR (DMSO-d6): δ 3.52
(s, 6H), 3.56 (s, 3H), 6.33 (s, 2H), 6.75−6.77 (m, 1H), 6.84−6.86 (m,
1H), 7.00−7.04 (m, 1H), 7.08−7.12 (m, 1H), 7.39 (d, J = 8.6 Hz,
2H), 7.48−7.50 (m, 1H), 11.11 (br s, 1H, disappeared on treatment
with D2O), 11.58 ppm (br s, 1H, disappeared on treatment with D2O).
MS: ES+ = 381 (MH+). IR: ν 3358, 3390 cm−1. Anal. (C21H20N2O3S
(380.46)) C, H, N, S.
2-(Thiophen-2-yl)-3-(3′,4′,5′-trimethoxybenzyl)-1H-indole
(11). A mixture of 10 (0.1 g, 0.00025 mol) and sodium borohydride
(0.1 g, 0.0025 mol) in ethanol (10 mL) was heated to reflux for 2.5 h.
After cooling, the reaction mixture was diluted with water and
extracted with ethyl acetate. The organic layer was washed with brine
and dried. Removal of the solvent gave a residue that was purified by
column chromatography (silica gel, ethyl acetate/n-hexane 3:7 as
eluent) to furnish 11 as a brown solid (0.02 g, 21%), mp 43−47 °C
(from ethanol). 1H NMR (CDCl3): δ 3.73 (s, 6H), 3.80 (s, 3H), 4.27
(s, 2H), 6.47 (s, 2H), 7.07−7.13 (m, 2H), 7.19−7.23 (m, 2H), 7.35−
7.39 (m, 2H), 7.49 (d, J = 7.9 Hz, 1H), 8.17 ppm (br s, 1H,
disappeared on treatment with D2O). MS: ES+ = 380 (MH+). IR: ν
3340 cm−1. Anal. (C22H21NO3S (379.47)) C, H, N, S.
2-(Furan-2-yl)-3-[(3′,4′,5′-trimethoxyphenyl)thio]-1H-indole
(6). Synthesized as 4, starting from 15, yield 38% as a white solid, mp
1
180−184 °C (from ethanol). H NMR (CDCl3): δ 3.65 (s, 6H), 3.77
(s, 3H), 6.38 (s, 2H), 6.54 (q, J = 1.8 Hz, 1H), 7.17−7.20 (m, 1H),
7.24−7.25 (m, 2H), 7.44 (d, J = 8 Hz, 1H), 7.50 (m, 1H), 7.67 (d, J =
8 Hz, 1H), 8.94 ppm (br s, 1H, disappeared on treatment with D2O).
MS: ES+ = 382 (MH+). IR: ν 3333 cm−1. Anal. (C21H19NO4S
(381.44)) C, H, N, S.
2-(Furan-3-yl)-3-[(3′,4′,5′-trimethoxyphenyl)thio]-1H-indole
(7). Synthesized as 4, starting from 16, yield 42% as a brown solid, mp
1
185−190 °C (from ethanol). H NMR (DMSO-d6): δ 3.64 (s, 6H),
2-(2-Nitrophenyl)-1-[1-(phenylsulfonyl)-1H-pyrrol-3-yl]-
ethan-1-one (21). Oxalyl chloride (2.46 g, 1.64 mL, 0.019 mol) and
a catalytic amount of anhydrous DMF were added at 0 °C to 20 (3.52
g, 0.019 mol) in anhydrous 1,2-dichloroethane (88 mL) under an Ar
stream. After 30 min, 1-(phenylsulfonyl)-1H-pyrrole (4.04 g, 0.0019
mol) and anhydrous aluminum chloride (2.59 g, 0.0019 mol) were
added. The reaction mixture was stirred at 0 °C for 25 min, quenched
on 1 N HCl/crushed ice, and extracted with chloroform. The organic
layer was washed with brine and dried. Removal of the solvent gave a
residue that was purified by column chromatography (silica gel,
acetone/n-hexane 1:2 as eluent) to furnish 21 as a yellow solid (0.7 g,
3.76 (s, 3H), 6.34 (s, 2H), 6.87 (m, 1H), 7.16 (t, J = 7.5 Hz, 1H),
7.22−7.26 (m, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.51 (t, J = 1.6 Hz, 1H),
7.65 (dd, J = 0.6 and 7.8 Hz, 1H), 8.08−8.09 (m, 1H), 8.50 ppm (br s,
1H, disappeared on treatment with D2O). MS: ES+ = 382 (MH+). IR:
ν 3330 cm−1. Anal. (C21H19NO4S (381.44)) C, H, N, S.
2-(Thiophen-2-yl)-3-[(3′,4′,5′-trimethoxyphenyl)thio]-1H-in-
dole (8). Synthesized as 4, starting from 17, yield 60% as a yellow
1
solid, mp 195−198 °C (from ethanol). H NMR (DMSO-d6): δ 3.53
(s, 6H), 3.56 (s, 3H), 6.35 (s, 2H), 7.09−7.13 (m, 1H), 7.19−7.24 (m,
2H), 7.47−7.49 (m, 2H), 7.65 (dd, J = 1.1 and 5.0 Hz, 1H), 7.77 (dd, J
= 1.1 and 3.7 Hz, 1H), 12.11 ppm (br s, 1H, disappeared on treatment
with D2O). MS: ES+ = 398 (MH+). IR: ν 3317 cm−1. Anal.
(C21H19NO3S2 (397.51)) C, H, N, S.
1
10%), mp 135 °C (from ethanol). H NMR (DMSO-d6): δ 4.45 (s,
2H), 6.41−6.43 (m, 1H), 7.25−7.30 (m, 2H), 7.45−7.60 (m, 5H),
7.85−7.86 (m, 1H), 7.90−7.92 (m, 2H), 8.06 ppm (dd, J = 1.2 and 8.2
Hz, 1H). IR: ν 1680 cm−1. Anal. (C18H14N2O5S (370.38)) C, H, N, S.
2-[1-(Phenylsulfonyl)-1H-pyrrol-3-yl]-1H-indole (14). A mix-
ture of 21 (0.25 g, 0.0007 mol) and iron powder (0.27 g, 0.0049 mol)
in glacial acetic acid (8 mL) was heated at 60 °C overnight. After
cooling, the mixture was diluted with water while stirring and extracted
with ethyl acetate. The organic layer was washed with brine and dried.
Removal of the solvent gave a residue that was purified by column
2-(Thiophen-3-yl)-3-[(3′,4′,5′-trimethoxyphenyl)thio]-1H-in-
dole (12). Synthesized as 4, starting from 19, yield 83% as a white
1
solid, mp 220−225 °C (from ethanol). H NMR (CDCl3): δ 3.64 (s,
6H), 3.78 (s, 3H), 6.37 (s, 2H), 7.17−7.21 (m, 1H), 7.25−7.29 (m,
1H), 7.42−7.44 (m, 2H), 7.64 (dd, J = 1.3 and 5.0 Hz, 1H), 7.68 (d, J
= 7.8 Hz, 1H), 7.85 (dd, J = 1.3 and 3.0 Hz, 1H), 8.62 ppm (br s, 1H,
disappeared on treatment with D2O). MS: ES+ = 398 (MH+). IR: ν
3331 cm−1. Anal. (C21H19NO3S2 (397.51)) C, H, N, S.
8402
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
chromatography (silica gel, ethyl acetate/n-hexane 1:3 as eluent) to
furnish 14 as a white solid (0.7 g, 10%), mp 180−185 °C (from
ethanol). 1H NMR (DMSO-d6): δ 6.66 (d, J = 1.4 Hz, 1H), 6.83−6.85
(m, 1H), 6.93−6.97 (m, 1H), 7.02−7.07 (m, 1H), 7.31 (d, J = 8.0 Hz,
1H), 7.44−7.46 (m, 2H), 7.65−7.69 (m, 2H), 7.74−7.79 (m, 1H),
7.84 (t, J = 1.9 Hz, 1H), 7.98−8.00 (m, 2H), 11.30 ppm (br s, 1H,
disappeared on treatment with D2O). IR: ν 3407 cm−1. Anal.
(C18H14N2O2S (322.38)) C, H, N, S.
1-(1-(Furan-2-yl)ethylidene)-2-phenylhydrazine (23). A mix-
ture of phenylhydrazine hydrochloride (3.91 g, 0.027 mol), 22 (2.0 g,
1.82 mL, 0.018 mol), and anhydrous sodium acetate (2.21 g, 0.027
mol) in ethanol (25 mL) was placed into the microwave cavity (open
vessel mode). Microwave irradiation of 250 W was used, the
temperature being ramped from 25 to 80 °C. Once 80 °C was
reached, taking about 1 min, the reaction mixture was held there for 5
min, while stirring and cooling. The reaction mixture was filtered to
give 23 as an orange solid (2.9 g, 96%), mp 79−84 °C (from ethanol).
1H NMR (DMSO-d6): δ 2.19 (s, 3H), 6.54−6.55 (m, 1H), 6.70−6.71
(m, 1H), 6.74−6.77 (m, 1H), 7.18−7.23 (m, 4H), 7.68−7.69 (m, 1H),
9.22 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν
3342 cm−1. Anal. (C12H12N2O2 (200.24)) C, H, N.
2-(Furan-2-yl)-1H-indole (15). 23 (1.18 g, 0.006 mol) was added
portionwise to PPA (10 g) preheated at 110 °C, and the reaction
mixture was stirred at the same temperature for 1 h. After quenching
on crushed ice, the mixture was stirred at 25 °C overnight; then it was
made basic (pH = 10) with a saturated solution of potassium
carbonate and extracted with ethyl acetate. The organic layer was
washed with brine and dried. Removal of the solvent gave a residue
that was purified by column chromatography (alumina, ethyl acetate/
n-hexane 1:5 as eluent) to furnish 15 as a yellow solid (0.6 g, 55%), mp
123−125 °C (from ethanol). Lit.29 120−123 °C.
3:7 as eluent) to furnish 17 as a yellow solid (0.19 g, 40%), mp 175−
180 °C (from cyclohexane). Lit.31 167−168 °C (from hexane).
2-Bromo-1-(thiophen-3-yl)ethanone (28). Synthesized as 26,
starting from 27. Yield 57% as a brown solid, mp 60−65 °C (from
ethanol). 1H NMR (CDCl3): δ 4.34 (s, 2H), 7.35−7.37 (m, 1H), 7.57
(dd, J = 1.3 and 5.1 Hz, 1H), 8.17−8.18 ppm (m, 1H). IR: ν 1672
cm−1. Anal. (C6H5BrOS (205.07)) C, H, Br, S.
2-(Thiophen-3-yl)-1H-indole (19). Synthesized as 17, starting
from 28. Yield 16% as a yellow solid, mp 210−215 °C (from
cyclohexane). Lit.32 212−214 °C.
2-(5-Methoxy-2-nitrophenyl)-1-(thiophen-2-yl)ethanol
(30). TBAF (1 M in THF, 0.83 mL, 0.00083 mol) was dropped into a
solution of 2926 (0.2 g, 0.00083 mol) and thiophen-2-carboxaldehyde
(0.11 g, 0.092 mL, 0.001 mol) in anhydrous THF (5 mL) at 25 °C
under an Ar stream. After 15 min, 37% HCl (0.83 mL) was added, and
the reaction mixture was extracted with diethyl ether, washed with
brine, and dried. Removal of the solvent gave a residue that was
purified by column chromatography (silica gel, chloroform as eluent)
1
to furnish 30 as a yellow oil (0.14 g, 61%). H NMR (CDCl3): δ 2.46
(d, J = 4.2 Hz, 1H), 3.37 (dd, J = 8.4 and 13.3 Hz, 1H), 3.55 (dd, J =
4.4 and 13.3 Hz, 1H), 3.84 (s, 3H), 5.31−5.35 (m, 1H), 6.77 (d, J =
2.8 Hz, 1H), 6.86 (dd, J = 2.8 and 9.1 Hz, 1H), 6.97−7.02 (m, 2H),
7.27−7.30 (m, 1H), 8.08 ppm (d, J = 9.1 Hz, 1H). IR: ν 3289 cm−1.
Anal. (C13H13NO4S (279.31)) C, H, N, S.
2-(5-Methoxy-2-nitrophenyl)-1-(thiophen-2-yl)ethan-1-one
(31). A solution of 30 (0.74 g, 0.00265 mol) in anhydrous
dichloromethane (1.7 mL) was dropped into a suspension of pyridium
chlorochromate (0.87 g, 0.004 mol) in the same solvent (6 mL). The
reaction mixture was stirred at 25 °C for 1.5 h and filtered. After
removal of the solvent, the crude product was purified by column
chromatography (silica gel, chloroform as eluent) to furnish 31 as a
1
white solid (0.7 g, 56%), mp 140−145 °C (from ethanol). H NMR
2-(Furan-3-yl)-1H-indole (16). A mixture of 2-iodo-1H-indole29
(0.25 g, 0.001 mol), furan-3-boronic acid pinacol ester (0.26 g,
0.00134 mol), potassium carbonate (0.18 g, 0.00134 mol), and Pd(II)
acetate (0.03 g, 0.000134 mol) in 1-methyl-2-pyrrolidinone (2.3 mL)
containing 0.18 mL of water was degassed for 15 min and placed into
the MW cavity (closed vessel mode, Pmax = 250 psi). MW irradiation
of 200 W was used, the temperature being ramped from 25 to 110 °C
while stirring. Once 110 °C was reached, taking about 2 min, the
reaction mixture was held there for 15 min. After cooling, the mixture
was diluted with water and extracted with ethyl acetate. The organic
layer was washed with brine and dried. Removal of the solvent gave a
residue that was purified by column chromatography (silica gel, ethyl
acetate/n-hexane 1:5 as eluent) to furnish 16 as a white solid (0.18 g,
(CDCl3): δ 3.90 (s, 3H), 4.65 (s, 2H), 6.83 (d, J = 2.7 Hz, 1H), 6.93
(dd, J = 2.8 and 9.1 Hz, 1H), 7.14−7.20 (m, 1H), 7.65−7.69 (m, 1H),
7.84−7.92 (m, 1H), 8.22 ppm (d, J = 9.2 Hz, 1H). IR: ν 1654 cm−1.
Anal. (C13H11NO4S (277.30)) C, H, N, S.
5-Methoxy-2-(thiophen-2-yl)-1H-indole (18). A mixture of 30
(1.07 g, 0.0039 mol) and tin(II) chloride dihydrate (4.98 g, 0.019 mol)
in ethyl acetate was heated to reflux for 3 h. After cooling, the reaction
mixture was made basic (pH = 10) with a saturated potassium
carbonate solution. The suspension was filtered and the layers were
separated. The organic layer was washed with brine and dried.
Removal of the solvent gave a residue that was purified by column
chromatography (silica gel, dichloromethane as eluent) to furnish 18
as a yellow solid (0.19 g, 21%), mp 125 °C (from ethanol). Lit.33 124
°C.
Biology. Tubulin Assembly. The reaction mixtures contained
0.8 M monosodium glutamate (pH 6.6 with HCl in a 2 M stock
solution), 10 μM tubulin, and varying concentrations of compound.
Following a 15 min preincubation at 30 °C, samples were chilled on
ice, GTP to 0.4 mM was added, and turbidity development was
followed at 350 nm in a temperature controlled recording
spectrophotometer for 20 min at 30 °C. Extent of reaction was
measured. Full experimental details were previously reported.34
[3H]Colchicine Binding Assay. The reaction mixtures contained
1.0 μM tubulin, 5.0 μM [3H]colchicine, and 5.0 μM inhibitor and were
incubated for 10 min at 37 °C. Complete details were described
previously.35
Cell Cultures and Treatment. Cell lines were obtained from the
American Tissue Culture Collection (ATCC), unless specified
otherwise. Cells were grown in Dulbecco’s Modified Eagle medium
(D-MEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C
with 5% CO2. In all experiments 300.000 cells were plated in 9 cm2
dishes and exposed to test compound dissolved in DMSO (0.1% final
concentration) at the indicated concentrations.
The methodology for the evaluation of the growth of human MCF-
7 breast carcinoma, OVCAR-8, and NCI/ADR-RES cells, obtained
from the National Cancer Institute drug screening laboratory, was
previously described, except that cells were grown for 96 h for IC50
determinations.46 HeLa, PC3, HT29, and A549 cell lines were grown
1
95%), mp 145−148 °C (from ethanol). H NMR (CDCl3): δ 6.61−
6.62 (m, 1H), 6.70−6.71 (m, 1H), 7.09−7.19 (m, 2H), 7.36 (dd, J =
0.9 and 8.0 Hz, 1H), 7.50−7.52 (m, 1H), 7.58−7.60 (m, 1H), 7.75 (m,
1H), 8.10 ppm (broad s, disappeared on treatment with D2O, 1H). IR:
ν 3417 cm−1. Anal. (C12H9NO (183.21)) C, H, N.
2-Bromo-1-(thiophen-2-yl)ethan-1-one (26). A solution of
bromine (2.53 g, 0.82 mL, 0.0158 mol) in dichloromethane (8 mL)
was added dropwise to 25 (2.0 g, 1.71 mL, 0.0158 mol) in the same
solvent (10 mL). The reaction mixture was stirred at 25 °C for 1 h and
neutralized with a saturated solution of sodium hydrogen carbonate.
The organic layer was washed with brine and dried. Removal of the
solvent gave a residue that was purified by column chromatography
(silica gel, ethyl acetate/n-hexane 5:95 as eluent) to furnish 26 as a
yellow oil (2.60 g, 80%).30
2-(Thiophen-2-yl)-1H-indole (17). A mixture of 26 (0.5 g,
0.0024 mol) and aniline (0.4 g, 0.4 mL, 0.0041 mol) was kept at 25 °C
for 3 h with occasional stirring; then a catalytic amount of anhydrous
DMF was added, and the reaction mixture was placed into the MW
cavity (closed vessel mode, Pmax = 250 PSI). MW irradiation of 150
W was used, the temperature being ramped from 25 to 100 °C while
stirring. Once 100 °C was reached, taking about 1 min, the reaction
mixture was held there for 1 min. The mixture was diluted with 1 N
HCl and extracted with ethyl acetate. The organic layer was washed
with brine and dried. Removal of the solvent gave a residue that was
purified by column chromatography (silica gel, ethyl acetate/n-hexane
8403
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
at 37 °C in D-MEM containing 10 mM glucose supplemented with
10% FBS, 100 units/mL each of penicillin and streptomycin, and 2
mM glutamine. At the onset of each experiment, cells were placed in
fresh medium and cultured in the presence of test compounds from
0.01 to 25 μM. 231-MDA and A549 cells were cultured in DMEM
supplemented with 10% FBS for 24, 48, and 72 h in a 96-well tissue
culture plate at 37 °C and 5% CO2 in the absence or presence of
different drug concentrations.
The Potorus tridactylis PtK2 cells, the A10 rat embryonic aortic
smooth muscle cells, the human umbilical vein endothelial cells, and
the human aortic smooth muscle cells were obtained from ATCC and
grown as recommended, except that a 5% CO2 atmosphere was used
with all cells.
Evaluation of growth inhibition of the A2780wt, A2780-CIS, and
OVCAR-3 cell lines was performed as previously reported.36
Cell Viability Assay. Cell viability was generally determined using
the MTT colorimetric assay. The test is based on the ability of
mitochondrial dehydrogenase to convert, in viable cells, the yellow
MTT reagent into a soluble blue formazan dye. Cells were seeded into
96-well plates to a density of 7 × 103/100 μL well. After 24 h of
growth to allow attachment to the wells, compounds were added at
various concentrations (from 0.01 to 25 μM). After 48 h of growth and
removal of the culture medium, 100 μL/well of medium containing 1
mg/mL of MTT was added. Cell cultures were further incubated at 37
°C for 2 h in the dark. The solution was then gently aspirated from
each well, and the formazan crystals within the cells were dissolved
with 100 μL of DMSO. Optical densities were read at 550 nm using a
Multiskan Spectrum Thermo Electron Corporation reader. Results
were expressed as percentage relative to vehicle-treated control (0.5%
DMSO was added to untreated cells), and IC50 values were
determined by linear and polynomial regression. Experiments were
performed in triplicate.
231-MDA and A549 cells were plated at different cellular densities
in order to test the compounds on logarithmic phase cells. Test
compounds were added after cell adhesion. On the day of the assay, 10
μL MTT (5 mg/mL) was added to each well, and cells were incubated
for 2 h (37 °C, 5% CO2). When dark crystals appeared at the well
bottom, culture medium was discarded, and ethanol (100 μL) was
added to each well to solubilize the crystals, yielding a purple solution.
Absorbance was read with an enzyme-linked immunosorbent assay
(ELISA) reader at 570 nm.
Immunofluoresence Assay. Cells to be processed for immuno-
fluoresence were grown on sterile poly-L-lysine (SIAL) coated
coverslips. After treatment, cells were either simultaneously fixed and
permeabilized in MeOH for 6 min at −20 °C or, alternatively, first
fixed in 3.7% paraformaldehyde (PFA) for 10 min followed by
permeabilization in 0.1% Triton-X 100. The following antibodies were
used: anti-β-tubulin unconjugated (Sigma clone B5.1.2, 1:2000) or
FITC-conjugated (Sigma, 1:150); anti-γ-tubulin (Sigma, 1:1000);
antiprocessed caspase-3 (Cell Signaling Technology, 9661, 1:500).
Antibodies attached to their antigens were detected using secondary
antibodies: antimouse Texas Red (Vector, TI-2000, 1:800), antimouse
FITC (Jackson Immunoresearch Laboratories, 115-095-068, 1:200),
antimouse AMCA (1:50), antirabbit FITC (Santa Cruz, sc-2090,
1:100), antirabbit Cy3 (Jackson Immunoresearch Laboratories, 711-
165-152, 1:1000). The DNA was stained with DAPI (Sigma, 0.05 μg/
mL in H2O). Slides were finally mounted in Vectashield (Vector).
Microscopy. Bright-field images of growing cells were taken under
an inverted fluorescence microscope (Nikon TE 300) equipped with a
DMX1200-type CCD (resolution 1280 pixels × 1024), using ACT-1
software. Immunofluoresence images were obtained under an
epifluorescence Olympus AX70 microscope equipped with a CCD
camera (Diagnostic Instruments, Spot RT slider model 2.3.0) or a
Nikon Eclipse 90i microscope equipped with Qimaging camera and
the NisElements AR 3.1 software (Nikon).
cytofluorometer (Beckman Coulter) equipped with EXPO 32 ADC
software. Data for at least 10000 cells per sample were acquired.
Western Blotting Analysis. Cells were plated in flasks (1 × 106
cells) and incubated with or without an ATI compound. At the
indicated times, cells were lysed using ice cold lysis buffer (50 mM
Tris, 150 mM NaCl, 10 mM EDTA, 1% Triton) supplemented with
protease inhibitor cocktail (antipain, bestatin, chymostatin, leupeptin,
pepstatin, phosphoramidon, Pefabloc, EDTA, and aprotinin, all from
Boehringer). Whole cell extracts were loaded on 8−12% sodium
dodecyl sulfate polyacrylamide gels and electrophoresed, followed by
blotting onto nitrocellulose membranes (BioRad). After membrane
blocking with 5% (w/v) fat-free milk powder, 0.1% Tween 20 in Tris
buffered saline (TBS), the membrane was incubated overnight at 4 °C
with specific antibodies at the concentrations indicated by the
manufacturers (Cell Signaling Technology and Santa Cruz Bio-
technology) in Tris-buffered saline/Tween-20/5% milk. Following
incubation with horseradish peroxidase-conjugated secondary anti-
body, bands were detected by enhanced chemiluminescence (ECL kit,
Amersham). Each filter was reprobed with mouse monoclonal
antiactin antibody. The signal intensity of detected bands was
quantified by NIH ImageJ 1.40 after normalization with actin.
Metabolic Stability. Test compounds in duplicate at the final
concentration of 1 μM were dissolved in DMSO and preincubated for
10 min at 37 °C in potassium phosphate buffer (pH 7.4) containing 3
mM MgCl2. The rat liver microsomes (Xenotech) were at a final
concentration of 0.5 mg/mL. After the preincubation period, reactions
were started by adding the cofactors mixture (NADP, glucose-6-
phosphate, glucose-6-phosphate dehydrogenase). Samples were taken
at times 0, 5, 10, 15, 20, and 30 min and added to acetonitrile to stop
the reaction, and the timed samples were centrifuged. Supernatants
were analyzed and quantified by LC-MS/MS. A control sample
without cofactors was always added in order to check the stability of
test compounds in the reaction mixtures. 7-Ethoxycoumarin and
propanolol were used as a reference standards. A fixed concentration
of verapamil was added in every sample as an internal standard for LC-
MS/MS.
Samples were analyzed on a UPLC (Waters) interfaced with a
Premiere XE Triple Quadrupole (Waters). Eluents were as follows:
phase A: 95% H2O, 5% acetonitrile + 0.1% HCOOH; phase B: 5%
H2O, 95% acetonitrile + 0.1% HCOOH. Flow rate, 0.6 mL/min;
column, Acquity BEH C18, 50 × 2.1 mm, 1.7 μm at 50 °C; injection
volume, 5 μL. Samples were analyzed under the following conditions:
electron spray ionization positive, desolvation temperature 450 °C,
capillary 3.5 kV, extractor 5 V. The percent of test compound
remaining after a 30 min incubation period was calculated from the
peak area relative to the area of the compound at time zero.
Aqueous Solubility. The solubility of compound 5 was measured
by means of the HTS. Samples were placed in a 96-well filter plate and
incubated at room temperature for 90 min. The plate was then filtered,
and solutions were analyzed by LC/MS-UV.
In Vivo Drug Pharmacokinetic Studies. Pharmacokinetic
experiments were performed using 4 week old male nude CD-1
mice (Charles River Laboratories). Animals were quarantined for
approximately 1 week prior to the study. They were housed under
standard conditions and had free access to water and a standard
laboratory rodent diet. Compound 5 was dissolved in a mixture of 3%
DMSO + 30% PEG400 + NaCl 0.9% at the concentration for iv (rapid
bolus) administration or a mixture of 5% DMSO + 20% PEG400 +
water for the oral (gavage) dose (dose volume 5 mL/kg). Compound
5 was administered to mice either by the iv or the oral route, and
blood samples were collected after different time points after dosing.
Plasma was separated immediately after blood sampling by
centrifugation, plasma proteins were precipitated using Sirocco
filtration plates or Oasis HLB elution plates according to the
distributor’s instructions, and the plasma samples were kept frozen
at −80 °C until LC/MS/MS analysis. Sample analysis was performed
on an Acquity UPLC using either a Acquity BEH C18 column (50 mm
× 2.1 mm × 1.7 μm) or a Acquity HSS T3 column (50 mm × 2.1 mm
× 1.8 μm), coupled with a sample organizer and interfaced to a triple
quadrupole Premiere XE (Waters). The mass spectrometer was
Flow Cytometry. Cell cycle distribution was analyzed after cell
incubation with propidum iodide (Sigma). Apoptosis was analyzed
after incubation of cells with annexin V-FITC (Immunofluorescence
Science). Cell samples were analyzed in a Coulter Epics XL
8404
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
operated using an electrospray interface (ESI) with a capillary voltage
of 3−4 kV, a cone voltage of 25−52 V, a source temperature of 115−
120 °C, a desolvation gas flow of 800 L/h, and a desolvation
temperature of 450−480 °C. The collision energy was optimized for
each compound. LC−MS/MS analyses were carried out using a
positive electrospray ionization interface in multiple reaction
monitoring mode. Pharmacokinetic parameters were calculated by a
noncompartmental method using WinNolin 5.1 software (Pharsight).
Statistical Analysis. Data were analyzed using Prism 4.0
(GraphPad Software, Inc.). Results are expressed as mean ± SEM.
binds covalently to microtubule pores and lumenal taxoid binding
sites. Nat. Chem. Biol. 2007, 3, 117−125.
(11) Sridhare, M.; Macapinlac, M. J.; Goel, S.; Verdier-Pinard, D.;
Fojo, T.; Rothenberg, M.; Colevas, D. The clinical development of
new mitotic inhibitors that stabilize the microtubule. Anticancer Drugs
2004, 15, 553−555.
(12) (a) Chen, S. M.; Meng, L. H.; Ding, J. New microtubule-
inhibiting anticancer agents. Exp. Opin. Invest. Drugs 2010, 19, 329−
343. (b) Harris, R.; Marx, G.; Gillett, M.; Kark, A.; Arunanthy, S.
Colchicine-induced bone marrow suppression: treatment with
granulocyte colony-stimulating factor. J. Emergency Med. 2000, 18,
435−440. (c) Mundy, W. R.; Tilson, H. A. Neurotoxic effects of
colchicine. Neurotoxicology 1990, 11, 539−547.
(13) Schmidt, M.; Bastians, H. Mitotic drug targets and the
development of novel anti-mitotic anticancer drugs. Drug Resist.
Update 2007, 10, 162−181.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional chemical and biological material. This material is
available free of charge via the Internet at http://pubs.acs.org.
(14) De Martino, G.; La Regina, G.; Coluccia, A.; Edler, M. C.;
Barbera, M. C.; Brancale, A.; Wilcox, E.; Hamel, E.; Artico, M.;
Silvestri, R. Arylthioindoles, potent inhibitors of tubulin
polymerization. J. Med. Chem. 2004, 47, 6120−6123.
AUTHOR INFORMATION
Corresponding Author
*Phone +39 06 4991 3800. Fax +39 06 4991 3133. E-mail:
romano.silvestri@uniroma1.it.
■
(15) De Martino, G.; Edler, M. C.; La Regina, G.; Coluccia, A.;
Barbera, M. C.; Barrow, D.; Nicholson, R. I.; Chiosis, G.; Brancale, A.;
Hamel, E.; Artico, M.; Silvestri, R. Arylthioindoles, potent inhibitors of
tubulin polymerization. 2. Structure activity relationships and
molecular modeling studies. J. Med. Chem. 2006, 49, 947−954.
(16) La Regina, G.; Edler, M. C.; Brancale, A.; Kandil, S.; Coluccia,
A.; Piscitelli, F.; Hamel, E.; De Martino, G.; Matesanz, R.; Díaz, J. F.;
Scovassi, A. I.; Prosperi, E.; Lavecchia, A.; Novellino, E.; Artico, M.;
Silvestri, R. New arythioindoles inhibitors of tubulin polymerization. 3.
Biological evaluation, SAR and molecular modeling studies. J. Med.
Chem. 2007, 50, 2865−2874.
(17) La Regina, G.; Sarkar, T.; Bai, R.; Edler, M. C.; Saletti, R.;
Coluccia, A.; Piscitelli, F.; Minelli, L.; Gatti, V.; Mazzoccoli, C.;
Palermo, V.; Mazzoni, C.; Falcone, C.; Scovassi, A. I.; Giansanti, V.;
Campiglia, P.; Porta, A.; Maresca, B.; Hamel, E.; Brancale, A.;
Novellino, E.; Silvestri, R. New arylthioindoles and related bioisosteres
at the sulfur bridging group. 4. Synthesis, tubulin polymerization, cell
growth inhibition, and molecular modeling studies. J. Med. Chem.
2009, 52, 7512−7527.
ACKNOWLEDGMENTS
This research was supported by PRIN 2008 Grant No.
200879X9N9, and Progetti di Ricerca di Universita,
■
̀
Sapienza
Universita
̀
di Roma. A.C. thanks Istituto PasteurFondazione
Cenci Bolognetti for his Borsa di Studio per Ricerche in Italia.
ABBREVIATIONS USED
■
MT, microtubule; ATI, arylthioindole; CSA4, combretastatin
A-4; VBL, vinblastine; PTX, paclitaxel; VBR, vinorelbine; SAR,
structure−activity relationship; MW, microwave; Pmax, max-
imum pressure; PPA, polyphosphoric acid; TBAF, tetrabuty-
lammonium fluoride; MI, mitotic index; DAMA-colchicine, N-
deacetyl-N-(2-mercaptoacetyl)colchicine; DAPI, 4′,6-diamidi-
no-2-phenylindole; Pgp, P-glycoprotein; FBS, fetal bovine
serum; PFA, paraformaldehyde
(18) Ciapetti, P.; Giethlen, B. Molecular variations based on isosteric
replacement. Carboxylic esters bioisosteres. In The practice of medicinal
chemistry, 3rd ed.; Wermuth, C. G., Elsevier Ltd: Oxford, U.K., 2008;
pp 310−313.
(19) Hugon, B.; Pfeiffer, B.; Renard, P.; Prudhomme, M. Synthesis of
isogranulatimide analogues possessing a pyrrole moiety instead of an
imidazole heterocycle. Tetrahedron Lett. 2003, 44, 3927−3930.
(20) Bergman, J.; Venemalm, L. Efficient synthesis of 2-chloro-, 2-
bromo-, and 2-iodoindole. J. Org. Chem. 1992, 57, 2495−2497.
(21) Bartoli, G.; Bosco, M.; Dalpozzo, R.; Todesco, P. E.
Functionalization of aromatic systems: a highly chemoselective
synthesis of trimethylsilyl-methylnitroarenes. J. Org. Chem. 1986, 51,
3694−3696.
REFERENCES
■
(1) Bhalla, K. N. Microtubule-targeted anticancer agents and
apoptosis. Oncogene 2003, 22, 9075−9086.
(2) Jordan, M. A.; Wilson, L. Microtubules as a target for anticancer
drugs. Nat. Rev. Cancer 2004, 4, 253−265.
(3) Teicher, B. A. Newer cytotoxic agents: attacking cancer broadly.
Clin. Cancer Res. 2008, 14, 1610−1617.
(4) Honore, S.; Pasquier, E.; Braguer, D. Understanding microtubule
dynamics for improved cancer therapy. Cell. Mol. Life Sci. 2005, 62,
3039−3056.
(5) Bhattacharyya, B.; Panda, D.; Gupta, S.; Banerjee, M. Anti-mitotic
activity of colchicine and the structural basis for its interaction with
tubulin. Med. Res. Rev. 2008, 28, 155−183.
(6) Ravelli, R. B.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.;
Sobel, A.; Knossow, M. Insight into tubulin regulation from a complex
with colchicine and a stathmin-like domain. Nature 2004, 428, 198−
202.
(7) Lin, M. C.; Ho, H. H.; Pettit, G. R.; Hamel, E. Antimitotic natural
products combretastatin A-4 and combretastatin A-2: studies on the
mechanism of their inhibition of the binding to colchicine to tubulin.
Biochemistry 1989, 28, 6984−6991.
(8) Nogales, E.; Whittaker, M.; Milligan, R. A.; Downing, K. H. High-
resolution model of the microtubule. Cell 1999, 96, 79−88.
(9) Nettles, J. H.; Li, H.; Cornett, B.; Krahn, J. M.; Snyder, J. P.;
Downing, K. H. The binding mode of epothilone A on alpha, beta-
tubulin by electron crystallography. Science 2004, 305, 866−869.
(10) Buey, R. M.; Calvo, E.; Barasoain, I.; Pineda, O.; Edler, M. C.;
Matesanz, R.; Cerezo, G.; Vanderwal, C. D.; Day, B. W.; Sorensen, E.
J.; Lopez, J. A.; Andreu, J. M.; Hamel, E.; Diaz, J. F. Cyclostreptin
(22) Shi, J.; Orth, J. D.; Mitchison, T. Cell type variation in responses
to antimitotic drugs that target microtubules and kinesin-5. Cancer Res.
2008, 68, 3269−3276.
(23) Gascoigne, K. E.; Taylor, S. S. Cancer cells display profound
intra- and interline variation following prolonged exposure to
antimitotic drugs. Cancer Cell 2008, 14, 111−122.
(24) Sudakin, V.; Yen, T. G. Targeting mitosis for anti-cancer
therapy. BioDrugs 2007, 21, 225−233.
(25) Gascoigne, K. E.; Taylor, S. S. How do anti-mitotic drugs kill
cancer cells? J. Cell Sci. 2009, 122, 2579−2585.
(26) Vitale, I.; Galluzzi, L.; Castedo, M.; Kroemer, G. Mitotic
catastrophe: a mechanism for avoiding genomic instability. Nat. Rev.
Mol. Cell. Biol. 2011, 12, 385−392.
(27) (a) Impens, F.; Van Damme, P.; Demol, H.; Van Damme, J.;
Vandekerckhove, J.; Gevaert, K. Mechanistic insight into taxol-induced
cell death. Oncogene 2008, 27, 4580−4591. (b) McGrogan, B. T.;
Gilmartin, B.; Carney, D. N.; McCann, A. Taxanes, microtubules and
8405
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406

Journal of Medicinal Chemistry
Article
chemoresistant breast cancer. Biochim. Biophys. Acta 2008, 2, 96−132.
(c) Mollinedo, F.; Gajate, C. Microtubules, microtubule-interfering
agents and apoptosis. Apoptosis 2003, 8, 413−450.
(28) Kerns, E. H.; Di, L. Plasma stability methods. In Drug-like
properties: concepts, structure design and methods; Academic Press:
Burlington, MA, 2008; pp 329−347.
(29) Kraus, G. A.; Guo, H. One-pot synthesis of 2-substituted indoles
from 2-aminobenzyl phosphonium salts. A formal total synthesis of
arcyriacyanin A. Org. Lett. 2008, 10, 3061−3063.
(30) An alternative preparation of 26 was recently described:
Ostrowski, T.; Golankiewicz, B.; De Clercq, E.; Andrei, G.; Snoeck, R.
Synthesis and anti-VZV activity of 6-heteroaryl derivatives of tricyclic
acyclovir and 9-{[cis-1′,2′-bis(hydroxymethyl)cycloprop-1′-
yl]methyl}guanine analogues. Eur. J. Med. Chem. 2009, 44, 3313−
3317.
(31) Hudkins, R. L.; Diebold, J. L.; Marsh, F. D. Synthesis of 2-aryl-
and 2-vinyl-1H-indoles via palladium-catalyzed cross-coupling of aryl
and vinyl halides with 1-carboxy-2-(tributylstannyl)indole. J. Org.
Chem. 1995, 60, 6218−6220.
(32) Fang, Y.-Q.; Lautens, M. A highly selective tandem cross-
coupling of gem-dihaloolefins for a modular, efficient synthesis of
highly functionalized indoles. J. Org. Chem. 2008, 73, 538−549.
(33) Caubere, C.; Caubere, P.; Ianelli, S.; Nardelli, M.; Jamart-
Gregoire, B. Aggregative activation and heterocyclic chemistry. I.
Complex base promoted arynic cyclization of imines or enamino
ketones; regiochemical synthesis of indoles. Tetrahedron 1994, 50,
11903−11920.
(34) Hamel, E. Evaluation of antimitotic agents by quantitative
comparisons of their effects on the polymerization of purified tubulin.
Cell Biochem. Biophys. 2003, 38, 1−21.
(35) Verdier-Pinard, P.; Lai, J.-Y.; Yoo, H.-D.; Yu, J.; Marquez, B.;
Nagle, D. G.; Nambu, M.; White, J. D.; Falck, J. R.; Gerwick, W. H.;
Day, B. W.; Hamel, E. Structure-activity analysis of the interaction of
curacin A, the potent colchicine site antimitotic agent, with tubulin and
effects of analogs on the growth of MCF-7 breast cancer cells. Mol.
Pharmacol. 1998, 35, 62−76.
(36) Ferlini, C.; Raspaglio, G.; Mozzetti, S.; Cicchillitti, L.; Filippetti,
F.; Gallo, D.; Fattorusso, C.; Campiani, G.; Scambia, G. The seco-
taxane IDN5390 is able to target class III β-tubulin and to overcome
paclitaxel resistance. Cancer Res. 2005, 65, 2397−2405.
8406
dx.doi.org/10.1021/jm2012886 | J. Med. Chem. 2011, 54, 8394−8406