New arylthioindoles and related bioisosteres at the sulfur bridging group. 4. Synthesis, tubulin polymerization, cell growth inhibition, and molecular modeling studies

Article abstract of DOI:10.1021/jm900016t

New arylthioindoles along with the corresponding ketone and methylene compounds were potent tubulin assembly inhibitors. As growth inhibitors of MCF-7 cells, sulfur derivatives were superior or sometimes equivalent to the ketones, while methylene derivatives were substantially less effective. Esters 24, 27-29, 36, 39, and 41 showed ～50% of inhibition on human HeLa and HCT116/chr3 cells at 0.5 μM, and these compounds inhibited the growth of HEK, M14, and U937 cells with IC50's in the 78-220 nM range. While murine macrophage J744.1 cell growth was significantly less affected (20% at higher concentrations), four other nontransformed cell lines remained sensitive to these esters. The effect of drug treatment on cell morphology was examined by time-lapse microscopy. In a protocol set up to evaluate toxicity on the Saccharomyces cerevisiae BY4741 wild type strain, compounds 24 and 54 strongly reduced cell growth, and 29, 36, and 39 also showed significant inhibition. 2009 American Chemical Society.

Full text of DOI:10.1021/jm900016t

7512 J. Med. Chem. 2009, 52, 7512–7527
DOI: 10.1021/jm900016t
New Arylthioindoles and Related Bioisosteres at the Sulfur Bridging Group. 4. Synthesis,
Tubulin Polymerization, Cell Growth Inhibition, and Molecular Modeling Studies
Giuseppe La Regina,† Taradas Sarkar,§ Ruoli Bai,§ Michael C. Edler,§ Roberto Saletti,‡ Antonio Coluccia,‡
Francesco Piscitelli,†,[ Lara Minelli,† Valerio Gatti,† Carmela Mazzoccoli,†,ꢀ Vanessa Palermo,# Cristina Mazzoni,#
Claudio Falcone,# Anna Ivana Scovassi,^ Vincenzo Giansanti,^ Pietro Campiglia,3 Amalia Porta,O Bruno Maresca,O
Ernest Hamel,§ Andrea Brancale,‡ Ettore Novellino, and Romano Silvestri*,†
†Istituto Pasteur;Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universitaꢀdi Roma, Piazzale Aldo
Moro 5, I-00185 Roma, Italy, ‡Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, U.K., §Toxicology
and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at
Frederick, National Institutes of Health, Frederick, Maryland 21702, Dipartimento di Chimica Farmaceutica e Tossicologica, Universitaꢀ di
Napoli Federico II, Via Domenico Montesano 49, I-80131, Napoli, Italy, ^Istituto di Genetica Molecolare;Consiglio Nazionale delle Ricerche,
Via Abbiategrasso 207, I-27100 Pavia, Italy, #Dipartimento di Biologia Cellulare e dello Sviluppo, Sapienza Universitaꢀ di Roma, Piazzale Aldo
Moro 5, I-00185 Roma, Italy, 3Universitaꢀ di Salerno, Dipartimento di Scienze Farmaceutiche, Sezione Chimico-Tecnologica, Via Ponte don
Melillo, I-84084 Fisciano, Salerno, Italy, and OUniversita di Salerno, Dipartimento di Scienze Farmaceutiche, Sezione Biomedica, Via Ponte don
ꢀ
Melillo, I-84084 Fisciano, Salerno, Italy. [ Current address: University of Pennsylvania, Department of Chemistry, 231 South 34th Street,
Philadelphia, PA 19104-6323. ꢀ Research project developed at the Istituto di Ricovero e Cura a Carattere Scientifico, Centro di Riferimento
Oncologico della Basilicata, Via Padre Pio 1, I-85028 Rionero in Vulture, Potenza, Italy.
Received January 8, 2009
New arylthioindoles along with the corresponding ketone and methylene compounds were potent
tubulin assembly inhibitors. As growth inhibitors of MCF-7 cells, sulfur derivatives were superior or
sometimes equivalent to the ketones, while methylene derivatives were substantially less effective. Esters
24, 27-29, 36, 39, and 41 showed ∼50% of inhibition on human HeLa and HCT116/chr3 cells at
0.5 μM, and these compounds inhibited the growth of HEK, M14, and U937 cells with IC50’s in the 78-
220 nM range. While murine macrophage J744.1 cell growth was significantly less affected (20% at
higher concentrations), four other nontransformed cell lines remained sensitive to these esters. The
effect of drug treatment on cell morphology was examined by time-lapse microscopy. In a protocol set
up to evaluate toxicity on the Saccharomyces cerevisiae BY4741 wild type strain, compounds 24 and 54
strongly reduced cell growth, and 29, 36, and 39 also showed significant inhibition.
Introduction
cellular apoptosis. Taxoids and epothilones target a lumenal
site on the β-subunit of dimers in microtubules4,5 and may
enterthe lumenthrougha binding site6 locatedataporeon the
microtubule surface formed by two different tubulin dimers.
Paclitaxel stimulates microtubule polymerization and stabili-
zation at high concentrations, whereas at lower concentra-
tions the drug inhibits microtubule dynamics with little effect
on the proportion of tubulin in polymer.7,8 Independent of
precise mechanism of action, clinical use of antitubulin drugs
is associated with problems of drug resistance, toxicity, and
bioavailability.9
Microtubules are a key target for anticancer agents.1 Tu-
bulin interacting agents interfere with the dynamic equilibri-
um of microtubules by either inhibition of tubulin
polymerization or blocking microtubule disassembly, and
both effects lead cells to the arrest of cell division. Colchicine
(1), combretastatin A-4 (CSA4,a 2), and the Catharanthus
alkaloids vincristine and vinblastine are tubulin assembly
inhibitors that interact with R,β-tubulin dimers at distinct
interfaces between the two subunits2,3 (Chart 1). These inter-
actions result in microtubule destabilization and subsequent
Arylthioindoles (ATIs) are potent tubulin assembly inhibi-
tors that bind to the colchicine site on β-tubulin close to its
interface with R-tubulin within the R,β-dimer.10 ATIs effi-
ciently inhibit tubulin polymerization and cancer cell growth,
with activities comparable with those of colchicine and CSA4.
To rationalize the structure-activity relationship (SAR) stu-
dies, we dissected the ATI structure into four regions: (A) the
substituent at position 2 of the indole, (B) the sulfur atom
bridge, (C) the 3-arylthio group, and (D) the substituent at
position 5 of the indole (Chart 1). In our previous papers, we
reported SAR and molecular modeling studies on ATI deri-
vatives differing in substituent groups and/or position at the
A, C, and D regions.11,12 However, chemical modification of
the sulfur bridge (B region) was not exhaustively explored,
*To whom correspondence should be addressed. Phone þ39 06 4991
3800. Fax: þ39 06 491 491. E-mail: romano.silvestri@uniroma1.it.
a Abbreviations: ATI, arylthioindole; CSA4, combretastatin A-4;
SAR, structure-activity relationship; MW, microwave; Pmax, maxi-
mum pressure; TMSDM, trimethylsilyldiazomethane; MCPBA, 3-
chloroperoxybenzoic acid; PPA, polyphosphoric acid; TBAHS, tetra-
butylammoniun hydrogen sulfate; DIPAD, diisopropyl azodicarboxy-
late; DMSO, dimethylsulfoxide; THF, tetrahydrofuran; MCF-7, human
breast carcinoma cells; HeLa, cervical cancer cells from Henrietta Lacks;
HCT116/chr3, human colon cancer cells; HEK, human embryonic
kidney 293 cells; M14, human melanoma cells; U937, human monocytic
leukemia cells; J774.1, murine macrophage cells; TLM, time-lapse
microscopy, a slow process technique of photographing, which allows
watching cellular growth; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide; PDB, protein data bank, DAMA-colchi-
cine, N-deacetyl-N-(2-mercaptoacetyl)colchicine.
r
pubs.acs.org/jmc
Published on Web 07/14/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7513
Chart 1. Structures of Derivatives 3-56 and Reference Compounds 1 and 2
Scheme 1. Synthesis of Compounds 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 25, 26, 28, 29, 31, 32, 34, 35, 39-41 and 43-45a
a Reagents and reaction conditions: (a) 3,4,5-trimethoxybenzoyl chloride, AlCl3, 1,2-dichloroethane, closed vessel, 110 ꢀC, 150 W, Pmax = 250 PSI,
2 min, 40-68%; (b) (5, 14, 26, and 35) NaBH4 (10 equiv), EtOH, reflux, 3 h, 42-68%; (c) (8, 11, 17, 20, 29, 32, 41, and 45) Et3SiH, CF3COOH, 25 ꢀC,
overnight, 46-90%; (d) (40 and 44) NaBH4 (1 equiv), THF/H2O, reflux, 2 h, 29-53%.
although five ATIs were transformed into the corresponding
sulfur dioxides.11 In contrast tothe weakpotency displayed by
these dioxides,11 several CSA4 analogues, characterized by a
carbonyl functionality as a bridging group between the indole
nucleus and the trimethoxyphenyl ring, show potent inhibi-
tion of tubulin polymerization.13 However, in comparing
ATIs with the 3-aroylindoles reported by Hsieh and co-work-
ers,14 we should note that these two groups of compounds are
significantly different because there are major differences in
our SAR findings and those of Hsieh’s group. Moreover, our
molecular modeling studies indicate that in the colchicine site
of tubulin, ATI derivatives preferentially assume a colchicine-
like rather than a CSA4-like binding conformation.10-12
These observations prompted the SAR investigations at the
B bridge reported here. To this end, we synthesized new indole
derivatives by bioisosteric replacement of the sulfur bridge of
the ATI’s with either carbonyl or methylene moieties, and
other related groups (compounds 3-45, Table 1). In addition,
we examined the replacement of the indole ring with differ-
ently substituted pyrrole and imidazole analogues
(compounds 46-56, Table 2).
Chemistry. Compounds 4, 7, 10, 13, 16, 19, 25, 28, 31, 34,
39, and 43 were synthesized in a microwave (MW) reactor by
treating the appropriate indole with 3,4,5-trimethoxyben-
zoyl chloride in the presence of aluminum chloride in 1,2-
dichloroethane at 110 ꢀC (150 W) for 2 min (Scheme 1).
Similarly, ethyl 5-chloro-3-[2-(3,4,5-trimethoxyphenyl)ace-
tyl]-1H-indole-2-carboxylate 21 was prepared by MW synth-
esis using ethyl 5-chloro-1H-indole-2-carboxylate and
2-(3,4,5-trimethoxyphenyl)acetyl chloride. The latter re-
agent was obtained by treating the appropriate carboxylic
acid with oxalyl chloride in the presence of a catalytic
amount of DMF in anhydrous THF at 0 ꢀC for 10 min
(Scheme 2). Sodium borohydride (10 equiv) reduction of 4,
13, 25, and 34 in boiling ethanol for 3 h furnished the
corresponding benzyl derivatives 5, 14, 26, and 35, respec-
tively (Scheme 1). Alternatively, benzyl (8, 11, 17, 20, 29, 32,
41, 45) and phenylethyl (23) derivatives were obtained in
good yield by treating benzoyl (7, 10, 16, 19, 28, 31, 39, and
43) (Scheme 1) or oxalyl (22) (Scheme 2) compounds with
triethylsilane in trifluoroacetic acid at 25 ꢀC for 12 h.
Reduction of ketones 39 and 43 with sodium borohydride
(1 equiv) in refluxing aqueous THF for 2 h gave the corre-
sponding alcohols 40 and 44. MW oxidation of ethyl
3-[2-(3,4,5-trimethoxyphenyl)acetyl]-1H-indole-2-carboxy-
late (21) with selenium(IV) oxide in dimethylsulfoxide
(DMSO) at 150 ꢀC (150 W) for 2 min furnished the corre-
sponding 2-oxo derivative 22.
Methyl (27) and ethyl (30) 5-bromo-3-(3,4,5-trimetho-
xyphenylthio)-1H-indole-2-carboxylate were obtained
by MW reaction of 5-bromo-1H-indole-2-carboxylic acid
(59) with bis-(3,4,5-trimethoxyphenyl)disulfide10 in the
presence of sodium hydride in anhydrous DMF at 110 ꢀC
(150 W) for 2 min (Scheme 3). The crude carboxylic acid
was transformed into the corresponding methyl ester 27
with trimethylsilyldiazomethane (TMSDM) in dichloro-
methane/methanol at 25 ꢀC for 30 min or into the ethyl
ester 30 with thionyl chloride in absolute ethanol at 65 ꢀC
for 2 h.
Oxidation of methyl 5-methoxy-3-(3,4,5-trimethoxyphe-
nylthio)-1H-indole-2-carboxylate (36)10 with 1 or 2 equiv of
3-chloroperoxybenzoic acid (MCPBA) in chloroform at

7514 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
La Regina et al.
Table 1. Inhibition of Tubulin Polymerization, Growth of MCF-7 Human Breast Carcinoma Cells and Colchicine Binding by Compounds 3-45 and
Reference Compound 2
compd
R1
R2
X
tubulina IC50 ( SD ( μM)
MCF-7b IC50 ( SD (nM)
inhibition colchicine bindingc (% ( SD)
3
H
H
H
H
H
H
H
H
H
H
H
H
S
2.6 ( 0.2
3.5 ( 0.07
28 ( 2
2.9 ( 0.1
2.7 ( 0.3
4.2 ( 0.02
2.9 ( 0.2
2.6 ( 0.3
5.4 ( 1
2.6 ( 0.2
2.5 ( 0.5
24 ( 2
2.5 ( 0.3
1.6 ( 0.1
1.7 ( 0.5
2.2 ( 0.2
1.4 ( 0.2
2.5 ( 0.5
>40
34 ( 9
150 ( 50
nd
68 ( 0.8
26 ( 0.3
nd
4
CdO
CH2
S
5
6
COOMe
COOMe
COOMe
COOEt
COOEt
COOEt
H
25 ( 1
33 ( 10
190 ( 70
40 ( 2
80 ( 20
nd
74 ( 2
49 ( 10
33 ( 7
19 ( 7
51 ( 6
nd
7
CdO
CH2
S
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
2
CdO
CH2
S
5-Cl
5-Cl
77 ( 7
53 ( 10
nd
51 ( 4
44 ( 9
nd
H
CdO
CH2
S
H
5-Cl
5-Cl
COOMe
COOMe
COOMe
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
H
42 ( 10
30 ( 6
87 ( 30
110 ( 2
110 ( 10
230 ( 60
nd
73 ( 0.2
61 ( 4
49 ( 4
53 ( 3
49 ( 3
39 ( 4
nd
5-Cl
5-Cl
CdO
CH2
S
5-Cl
5-Cl
CdO
CH2
COCH2
COCO
CH2CH2
S
5-Cl
5-Cl
5-Cl
5-Cl
>40
>40
nd
nd
nd
nd
5-Br
5-Br
1.6 ( 0.3
1.9 ( 0.3
13 ( 0.8
0.99 ( 0.1
1.3 ( 0.08
1.3 ( 0.08
1.6 ( 0.2
1.6 ( 0.05
1.7 ( 0.2
4.1 ( 0.6
3.4 ( 0.4
14 ( 0.5
2.0 ( 0.2
>40
43 ( 7
60d
nd
65 ( 3
45 ( 5
nd
H
CdO
CH2
S
H
5-Br
5-Br
COOMe
COOMe
COOMe
COOEt
COOEt
COOEt
H
33 ( 10
18 ( 4
30 ( 9
83 ( 20
67 ( 10
100d
75 ( 3
67 ( 4
59 ( 7
62 ( 7
58 ( 2
53 ( 4
61 ( 4
32 ( 7
nd
5-Br
5-Br
CdO
CH2
S
5-Br
5-Br
CdO
CH2
S
5-Br
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
22 ( 2
200d
nd
H
CdO
CH2
S
H
COOMe
COOMe
COOMe
COOMe
COOMe
COOMe
COOEt
COOEt
COOEt
COOEt
13 ( 3
nd
nd
93 ( 0.8
nd
nd
SdO
S(=O)2
CdO
CHOH
CH2
S
>40
0.67 ( 0.02
>40
17 ( 6
nd
78 ( 6
nd
1.4 ( 0.2
2.4 ( 0.2
2.6 ( 0.04
>40
33 ( 10
46 ( 3
90 ( 10
nd
59 ( 1
71 ( 2
49 ( 4
nd
CdO
CHOH
CH2
2.8 ( 0.3
1.1 ( 0.1
93 ( 10
2.5 ( 0.6
42 ( 4
99 ( 0.7
a Inhibition of tubulin polymerization. Tubulin was 10 μM during polymerization. b Inhibition of growth of MCF-7 human breast carcinoma cells.
c Inhibition of [3H]colchicine binding. Tubulin was at 1 μM, both [3H]colchicine and inhibitor were at 5 μM. d Same value obtained in all experiments.
25 ꢀC for 1.5 h gave the corresponding sulfoxide (37) or
sulfone (38) derivatives, respectively (Scheme 4). Ethyl 5-
bromo-1H-indole-2-carboxylate (59) was prepared by intra-
molecular cyclization of the corresponding ethyl pyruvate 4-
bromophenylhydrazone (57) in polyphosphoric acid (PPA)
as a catalyst, according to Fischer’s indole synthesis. The
needed hydrazone 57 was obtained by MW reaction of
4-bromophenylhydrazine hydrochloride with ethyl pyruvate
and sodium acetate in ethanol at 100 ꢀC (250 W) for 5 min.
MW hydrolysis of ester 58 with 3 N sodium hydroxide at
110 ꢀC (150 W) for 1 min furnished 5-bromo-1H-indole-
2-carboxylic acid (59), which was transformed into
the corresponding methyl ester 50 by treatment with
thionyl chloride in anhydrous methanol at 65 ꢀC for 2 h
(Scheme 5).
Compounds 46-49 were synthesized by phase-transfer reac-
tion from an appropriate ethyl 1H-pyrrole-2-carboxylate and
3,4,5-trimethoxybenzyl chloride15 in the presence of tetrabuty-
lammoniun hydrogen sulfate (TBAHS) in 50% sodium
hydroxide/dichloromethane. Reaction of 3,4,5-trimethoxyben-
zyl alcohol with ethyl 1H-imidazole-4-carboxylate in the
presence of diisopropyl azodicarboxylate (DIPAD) according

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7515
Table 2. Inhibition of Tubulin Polymerization, Growth of MCF-7 Human Breast Carcinoma Cells by Compounds 46-56, and Reference Compound 2
compd
R1
R2
R3
X
Y
tubulina IC50 ( SD (μM)
MCF-7b IC50 ( SD (nM)
46
47
48
49
50
51
52
53
54
55
56
2
COOEt
COOEt
COOEt
COOEt
H
H
H
H
CH2
CH2
CH2
CH2
CH2
CdO
CdO
CdO
CH2
CH2
CH2
CH
>40
>40
nd
nd
Me
Me
Me
H
C-Me
C-Me
C-Et
N
Me
Et
>40
>40
nd
nd
COOEt
H
>40
nd
COOEt
H
H
CH
N
32 ( 0.6
>40
>40
>2500
>2500
>2500
>2500
nd
COOEt
H
H
H
COOH
COOEt
H
N
H
Me
CH
C-Me
N HCl
>40
>40
COO(CH2)2O(CH2)2OH
H
Me
H
COOH
>40
1.1 ( 0.1
nd
2.5 ( 0.6
3
a Inhibition of tubulin polymerization. Tubulin was 10 μM during polymerization. b Inhibition of growth of MCF-7 human breast carcinoma cells.
Scheme 2. Synthesis of Compounds 21, 22, and 23a
a Reagents and reaction conditions: (a) oxalyl chloride, anhydrous DMF cat., anhydrous THF, 0 ꢀC, 10 min; (b) ethyl 5-chloro-1H-indole-2-
carboxylate, AlCl3, 1,2-dichloroethane, closed vessel, 110 ꢀC, 150 W, Pmax = 250 PSI, 2 min, 32%; (c) selenium(IV) oxide, DMSO, closed vessel, 150 ꢀC,
150 W, Pmax = 250 PSI, 2 min, 56%; (d) Et3SiH, CF3COOH, 25 ꢀC, overnight, 40%.
Scheme 3. Synthesis of Compounds 27 and 30a
a Reagents and reaction conditions: (a) NaH, bis(3,4,5-trimethoxyphenyl)disulfide, anhydrous DMF, closed vessel, 110 ꢀC, 150 W, Pmax = 250 PSI,
PowerMAX, 2 min; (b) (27) TMSDM, CH2Cl2/MeOH, 25 ꢀC, 30 min, 79%; (c) (30) SOCl2, absolute EtOH, 65 ꢀC, 2 h, Ar stream, 70%.
to the Mitsunobu reaction16 afforded ethyl 1-(3,4,5-tri-
methoxybenzyl)-1H-imidazole-4-carboxylate (50). Reaction
of 3,4,5-trimethoxybenzoyl chloride with ethyl 1H-pyrrole-2-
carboxylate in the presence of potassium tert-butoxide and 18-
crown-6 or with ethyl 1H-imidazole-4-carboxylate gave ethyl
1-(3,4,5-trimethoxybenzoyl)-1H-pyrrole-2-carboxylate (51) or

7516 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
La Regina et al.
ethyl 1-(3,4,5-trimethoxybenzoyl)-1H-imidazole-4-carboxylate
(52) and ethyl 1-(3,4,5-trimethoxybenzoyl)-1H-imidazole-5-car-
boxylate (53), respectively. The esters 46 and 50 were hydrolyzed
into the corresponding carboxylic acids (54 and 56) by reacting
the appropriate ester with potassium hydroxide in aqueous
methanol or 37% hydrochloric acid, respectively. Ester 48 was
treated with potassium hydroxide in diethylene glycol at 140 ꢀC
for 3 h to obtain the corresponding carboxylic acid, which
underwent a transesterification reaction to furnish 2-(2-hydro-
xyethoxy)ethyl 3,4,5-trimethyl-1-(3,4,5-trimethoxybenzyl)-1H-
pyrrole-2-carboxylate (55) (Scheme 6). Attempts to obtain
3-(3,4,5-trimethoxybenzoyl)-1H-pyrrole by Friedel-Crafts acy-
lation of 1-tosyl-1H-pyrrole with 3,4,5-trimethoxybenzoyl chlor-
ide in the presence of anhydrous aluminum chloride (2 equiv)
invariably furnished (4-hydroxy-3,5-dimethoxyphenyl)-(1-tosyl-
1H-pyrrol-3-yl)methanone (scheme not shown).
Results and Discussion
Biological data for inhibition of tubulin polymerization,
binding of [3H]colchicine to tubulin (more active compounds
only), and growth of MCF-7 human breast carcinoma cells by
arylthioindoles 3-45 and reference compound 2 are summar-
ized in Table 1. MCF-7 cell growth was quantitated by
measuring cell protein with the dye sulforhodamine B after
48 h in the presence of various compound concentrations.
With few exceptions, tested derivatives showed potent
tubulin polymerization inhibitory activity. Replacement of
the sulfur atom with a carbonyl functionality led to com-
pounds endowed with comparable ability to inhibit tubulin
assembly (compare 3 with 4, 6 with 7, 9 with 10, 12 with 13, 15
with 16, 18 with 19, 24 with 25, 27 with 28, 30 with 31, 33 with
34, 36 with 39, and 42 with 43). Replacement of the carbonyl
with a methylene group led to inhibitors of tubulin assembly
whose potency was dependent on the substituent at position
2 of the indole. In particular, methylene compounds bearing a
methoxy- or ethoxycarbonyl group at this position differed
Scheme 4. Synthesis of compounds 37 and 38a
a Reagents and reaction conditions: (a) (37) MCPBA (1 equiv),
CHCl3, 25 ꢀC, 1.5 h, 99%; (b) (38) MCPBA (2 equiv), CHCl3, 25 ꢀC,
1.5 h, 96%.
Scheme 5. Synthesis of compounds 57-60a
a Reagents and reaction conditions: (a) ethyl pyruvate, CH3COONa, EtOH, open vessel, 100 ꢀC, 250 W, Pmax, 5 min, 90%; (b) PPA, 110 ꢀC, 30 min,
60%; (c) 3 N NaOH, closed vessel, 110 ꢀC, 150 W, 1 min, 95%; (d) SOCl2, absolute MeOH, 65 ꢀC, 2 h, Ar stream, 96%.
Scheme 6. Synthesis of Compounds 46-56a
a Reagents and reaction conditions: (a) (46-49, X =H2,Y =Cl) appropriate ethyl 1H-pyrrole-2-carboxylate, 3,4,5-trimethoxybenzyl chloride,
TBAHS, 50% KOH/DCM, 25 ꢀC, overnight, 45-57%; (b) (50, X=H2,Y=OH) ethyl 1H-imidazole-4-carboxylate, 3,4,5-trimethoxybenzyl alcohol,
P(Ph3)3, DIPAD, anhydrous THF, 25 ꢀC, overnight, Ar stream, 10%; (c) (51, X=O,Y=Cl) ethyl 1H-pyrrole-2-carboxylate, 3,4,5-trimethoxybenzoyl
chloride, 18-crown-6, t-BuOK, anhydrous THF, 25 ꢀC, 4 h, 8%; (d) (52 and 53, X = O,Y = Cl) ethyl 1H-imidazole-4-carboxylate, 3,4,5-
trimethoxybenzoyl chloride, anhydrous DCM, reflux temperature, 2.5 h, 13-15%; (e) (54) KOH, MeOH/H2O, reflux temperature, 3 h, 74%; (f )
(55) diethylene glycol, KOH, 140 ꢀC, 3 h, 34%; (g) (56) 37% HCl, reflux temperature, 1.5 h, 68%.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7517
little in activities from the corresponding sulfur/carbonyl
compounds (compare 8 with 6 and 7, 11 with 9 and 10, 17
with15 and 16, 20with18and 19, 29 with27and 28, 32with30
and 31, 41 with 39 and 36, and 45 with 43 and 42). In contrast,
when there was a hydrogen atom at position 2, there was a 3-
11-fold reduction in inhibitory activity (compare 5 with 3 and
4, 14 with 12 and 13, 26 with 24 and 25, and 35 with 33 and 34).
Seven compounds (21-23, 37, 38, 40, and 44) were completely
inactive. These results clearly indicate forbidden chemical
modifications at the B region, namely (i) elongation of the
bridging group from 1 to 2 atomic units (compounds 21-23),
(ii) oxidation of the sulfur atom to oxide (37) or dioxide
(37 and 38), as we also observed in our previous paper,11 and
(iii) reduction of the carbonyl bridge to an alcohol (40 and 44).
As growth inhibitors of MCF-7 human breast carcinoma
cells, the sulfur derivatives were superior (compare 3 with 4, 9
with 10, 24 with 25, 33 with 34, and 42 with 43) or equivalent
(compare 6 with 7, 12 with 13, 15 with 16, 18 with 19, 27 with
28, 30 with 31, and 36 with 39) to the corresponding ketones,
while methylene derivatives were usually less effective
(compare 8 with 6 and 7, 17 with 15 and 16, 20 with 18 and
19, 29 with27and 28, 32with30 and31, 41with39and 36, and
45 with 42 and 53). (Generally, only inhibitors of tubulin
polymerizationwithIC50<5 μM were evaluated for inhibition
of MCF-7 cell growth). The greatest inhibitory effects on
MCF-7 cells were observed with either sulfur or carbonyl
derivatives bearing a 2-methoxycarbonyl group at position
2 of the indole.
Table 3. Effects of 24, 27-29, 36, 39, and 41 on HeLa and HCT116/chr3
Cell Proliferation
% of cell proliferationa,b
HeLac
HCT116/chr3d
compd
0.5 μM
1 μM
0.5 μM
1 μM
24
27
28
29
36
39
41
47 ( 0.5
53 ( 0.1
41 ( 1.1
52 ( 0.4
57 ( 1.4
55 ( 0.7
52 ( 0.7
56 ( 1.2
60 ( 0.1
47 ( 0.4
45 ( 1.6
43 ( 0.3
54 ( 0.3
51 ( 0.2
60 ( 0.8
57 ( 0.9
59 ( 0.2
51 ( 4.6
63 ( 1.0
57 ( 3.4
69 ( 6.7
60 ( 3.9
62 ( 1.9
66 ( 3.5
51 ( 1.6
54 ( 1.0
55 ( 1.2
69 ( 0.3
a Data are expressed as % mean values ( SD; control cells are
considered as 100% (MTT method). b Treatments were performed for
24 h at the indicated concentrations. c HeLa cervical cancer cells.
d HCT116/chr3 human colon cancer cells.
Table 4. Inhibition of Growth of HEK, M14 and U937 Cells by
Compounds 24, 27-29, 36, 39, and 41a
IC50 ( SD (nM)
compd
HEKb
M14c
U937d
24
27
28
29
36
39
41
220 ( 3
189 ( 5
131 ( 3
160 ( 5
128 ( 7
140 ( 1
181 ( 3
194 ( 3
155 ( 9
78 ( 3
120 ( 10
111 ( 6
100 ( 5
137 ( 5
100 ( 3
177 ( 10
191 ( 7
159 ( 10
122 ( 6
155 ( 10
189 ( 5
a Growth inhibition of the indicated cell lines (MTT method); in-
cubation time was 48 h. b HEK, human embryonic kidney 293 cells;
c M14, human melanoma cells; d U937, human leukemic monocyte
lymphoma cells.
Compounds bearing either a bromine atom or a methoxy
group at position 5 and a 2-methoxycarbonyl group at posi-
tion 2 of the indole were found to be potent inhibitors in both
the tubulin polymerization and MCF-7 cell growth assays,
with potencies comparable to those of reference compound 2.
Worthy of note, among these esters, the methylene derivatives
29 and 41 were also highly active in both assays. In previous
studies, ATIs showed comparable11,12 to greater10 inhibition
of tubulin polymerizarion than 1 and adopted a binding
orientation similar to that of DAMA-colchicine.10
Compounds that inhibited tubulin assembly with IC50’s <
5 μM were also evaluated for inhibition of the binding of
[3H]colchicine to tubulin. Although none was as potent as
CSA4, significant inhibition was observed with all active
agents. As usually occurs with a series of antitubulin com-
pounds, there was not a linear correlation between colchicine
binding inhibition and inhibition of tubulin assembly. The
most active inhibitor of colchicine binding was compound 36,
which was almost as active as CSA4, although the difference
was more noticeable when the inhibitor concentration was
1 μM instead of 5 μM (data not presented). Compound 36 was
about half as active as CSA4 as an inhibitor of assembly
(IC50’s of 2.0 and 1.1 μM, respectively). Compounds 27 and
39, both more active than CSA4 as inhibitors of assembly
(IC50’s of 0.99 and 0.67μM, respectively), were less activethan
36 as inhibitors of colchicine binding.
findings is suggested in the modeling section (see below).
Surprisingly, compound54, which was inactiveagainstbovine
tubulin and MCF-7 cells, effectively inhibited growth of the
Saccharomyces cerevisiae BY4741 strain. Perhaps 54 might
interact with yeast tubulin, and this hypothesis is under
investigation in our laboratories.
The potent esters 24, 27-29, 36, 39, and 41 were evaluated
at 0.5 and 1.0 μM for cell growth inhibition on human HeLa
and HCT116/chr3 cells, derived from a cervix uterine carci-
noma and a colon carcinoma, respectively. All compounds
caused about 50% growth inhibition at the lower concentra-
tion tested (Table 3).
Compounds 24, 27-29, 36, 39, and 41 also reduced the
viability of HEK, M14, and U937 cells in a dose- and time-
dependent manner with IC50 values ranging from 78 nM (28,
M14 cells) to 220 nM (24, HEK cells) (Table 4). In contrast, at
300 nM, these compounds caused only 20% reduction in
murine macrophage J744.1 cell growth compared with un-
treated controls (Figure 1S, Supporting Information). Thus
these compounds were active in six cancer cell lines, but they
showed a reduced cytototoxic effect on the nonmalignant
murine macrophage J744.1 cells.
This observation with the J744.1 cells raised the possibility
that there might be a differential effect of esters 24, 27-29, 36,
39, and 41 in malignant and nontransformed cells, which in
turn would suggest the possibility of a potential improvement
in the therapeutic index for the ATI’s versus other antitubulin
agents. Accordingly, we expanded our study of nontrans-
formed cells to an epithelial line (PtK2, Potorus tridactylis
kidney epithelial cells), two aortic smooth muscle lines
(human and rat), and an endothelial line (human umbilical
We designed derivatives 46-56 for correlation with the
structure of the ATI’s by replacing the indole ring with
differently substituted pyrrole and imidazole analogues
(Table 2). Predictive docking studies suggested the synthesis
of 46-56 because we observed a close overlap between their
trimethoxyphenyl group and the corresponding ring A of the
cocrystallized DAMA-colchicine in the colchicine site on
tubulin. However, only 51 showed any inhibition of tubulin
polymerization and, in keeping with its weak activity, 51 did
not inhibit MCF-7 cell growth. An explanation of these

7518 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
La Regina et al.
Table 5. Inhibition of Growth and MCF-7 Selectivity Index of HAOSMC, PtK2, A10, and HUVEC Cells by Compounds 24, 27-29, 36, 39, and 41 and
Reference Compounds 2 and Paclitaxela
IC50 ( SD (nM)
compd
MCF-7
HAOSMCb (MCF-7 SI)f
PtK2c (MCF-7 SI)
A10d (MCF-7 SI)
HUVECe (MCF-7 SI)
24
43 ( 7
300 ( 200
(7.0)
230 ( 100
(5.3)
210 ( 100
(4.9)
400 ( 100
(9.3)
27
28
29
36
39
41
2
33 ( 10
18 ( 4
100 ( 30
(3.0)
280 ( 30
(8.5)
230 ( 100
(7.0)
140 ( 20
(4.2)
55 ( 40
(3.1)
110 ( 60
(6.1)
280 ( 40
(16)
150 ( 0g
(8.3)
30 ( 9
35 ( 7
(1.2)
270 ( 100
(9.0)
230 ( 100
(7.7)
140 ( 20
(4.7)
13 ( 3
15 ( 7 4
(1.2)
78 ( 50
(6.0)
70 ( 70
(5.4)
38 ( 30
(2.9)
17 ( 6
35 ( 7
(2.1)
190 ( 20
(11)
95 ( 80
(5.6)
110 ( 60
(6.5)
33 ( 10
2.5 ( 0.6
3.0 ( 0.5
70 ( 10
(2.1)
190 ( 200
(5.8)
130 ( 100
(3.9)
170 ( 50
(5.2)
18 ( 4
(7.2)
1.0 ( 1
(0.4)
23 ( 10
(9.2)
5.0 ( 5
(2.0)
Ptxh
6.0 ( 6
(2.0)
21 ( 8
(7.0)
38 ( 10
(13)
7.0 ( 4
(2.3)
a Growth inhibition of the indicated cell lines. b HAOSMC: human aortic smooth muscle cells (ATCC CLR-1999). c PtK2: potorous tridactylis kidney
epithelial cells (ATCC CLL-56). d A10, rat embryonic aortic smooth muscle cells (ATCC CRL-1476). e HUVEC: human umbilical vein endothelial cells
(ATCC CRL-2873). f MCF-7 SI: selectivity index (SI) for each agent in each cell line versus the MCF-7 cells (IC50 in specific cell line divided by IC50 in
MCF-7 cells; the higher the SI, the more resistant the nontransformed cell line relative to the MCF-7 cells). g Same value obtained in both experiments.
h Ptx: paclitaxel.
vein endothelial cells). We also determined the effects of CSA4
and paclitaxel on the growth of these four lines (Table 5).
In all cases, except for compound 24 with the umbilical vein
endothelial cells, the ATI’s were more inhibitory than they
had been with the J744.1 cells (that is, all IC50’s were less than
300 nM). The cytotoxicity pattern with each agent was
different, although compound 36 was the most cytotoxic
and compound 24 was overall the least cytotoxic with these
four nontransformed cell lines. We devised the MCF-7 SI as a
test for the ideathatthe ATI’s wouldhaveselectivetoxicityfor
a tumor cell line as compared with nonmalignant cells and as
compared with previously described antitubulin agents. This
hope, raised with the J744.1 macrophage cells, was not
confirmed with the additional cell lines. The average SI was
4.8 with CSA4 and 6.1 with paclitaxel, although even with
these two agents specific SI’s differed from each other (the
kidney epithelial cells were highly sensitive to CSA4, while
both muscle lines were relatively resistant; with paclitaxel, the
human muscle cells were most sensitive, the rat muscle cells
most resistant). The average SI with the esters ranged from 4.3
with compound 41 to 7.3 with compound 28. We conclude
that there is no significant difference between compounds 24,
27-29, 36, 39, and 41 as compared with CSA4 and paclitaxel.
The effect of drug treatment on cell morphology was
examined by time-lapse microscopy (TLM) using a Leica
CTR6500 microscope. Cells were kept at 37 ꢀC under a 5%
carbon dioxide atmosphere for 48 h. The cytotoxic effects of
compounds 24, 27-29, 36, 39, and 41 on HEK and M14 cell
growth increased in a concentration- and time-dependent
manner. Figure 1 shows the effects of 24, 39, and 41 after a
48 h treatment. Control HEK and M14 cell cultures displayed
elongated bipolar or polygonal morphology, while a signifi-
canteffect oncellmorphologyand12-30% growthinhibition
was already observed asearlyas12 h aftercompound addition
(data not shown). The treatment for 48 h with 24, 39, or 41
induced significant morphologic changes in both cell lines,
which became rounded and developed large vacuoles. More-
over, cells of both lines were less elongated than the untreated
controls, and some cells that became rounded also had a
tendency to detach from the substrate. Similar changes were
observed after treatment with27-29 and 36 (datanot shown).
Saccharomyces cerevisiae budding yeast has been used to
enhance understanding of fundamental cellular and molecu-
lar processes occurring in mammalian cells, including DNA
replication, DNA recombination, cell division, protein turn-
over, vesicular trafficking, and mechanisms involved in long-
evity of cell life and cell death. Approximately 31% of yeast
genes have a mammalian homologue, and an additional 30%
of yeast genes show domain similarity.17 Potentially, yeast can
be a powerful model for the development of cell death-
directed drugs. For example, paclitaxel, arsenic, bleomycin,
and valproate induce apoptotic phenotypes in yeast.18 Yeast
has increased our understanding of the pathogenic role of
human proteins in neurodegenerative diseases.19 Finally,
Cassidy-Stone and co-workers20 identified MDIVI-1
(mitochondrial division inhibitor-1) by yeast screens of che-
mical libraries.
Here we set up a protocol to evaluate the toxicity of
compounds 24, 27, 28, and 54 on BY4741, a standard
laboratory wild type strain of S. cerevisiae. As shown in
Figure 2, 24 and 54 strongly reduced the viability of the
BY4741 strain cells, which were cell growth limited at the first
dilution (the second showed limited cell growth). Compound
29 also showed significant BY4741 inhibition (cell growth was
limited to the third dilution).
Despite the small test set used in this experiment, the ester
functionality of 27-29 clearly caused a drop in activity as
compared with the most potent compound24 lacking the ester
group. Even more striking is the contrast between the high
activity of 54 with the yeast cells and its inactivity in the
mammalian assays. Despite its inactivity with mammalian
tubulin, it is unclear from docking studies why 54 does not
bind in the colchicine site (see below). The biological behavior
of 54 in yeast may indicate an interaction with fungal tubulin,
or the activity of the compound in yeast may indicate an
alternate target (manuscript in preparation).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7519
Figure 1. Effects of compounds 24, 39, and 41 on HEK (top) and M14 (bottom) cell morphology and growth as seen by TLM (magnifi-
cation 10ꢀ).
Binding of Compounds to β-Tubulin in the Colchicine Site.
On the basis of our previous modeling results,10-12 we
performed a series of docking simulations on a virtual library
of structures with different bridges (B) in order to identify the
most promising compounds before their actual synthesis. We
also investigated in silico the potential of replacing the indole
ring with smaller heterocycles such as pyrrole or imidazole.
The results obtained for the indole series showed that
replacement of the sulfur atom in the bridge (B) with a
methylene or carbonyl moiety led to virtually identical dock-
ing poses as reported previously for ATIs. In particular, the
trimethoxyphenyl moiety was situated in close proximity to
Cys241, and the indole NH formed a hydrogen bond with
Thr179 (Figure 3). These results predicted that these bridge
modifications would lead to strong biological activity, and
this was confirmed by the experimental results. Conversely,
results for compounds with a two-carbon bridge were dif-
ferent. Such structures could not be correctly placed in the

7520 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
La Regina et al.
colchicine site. The trimethoxyphenyl ring was more distant
from Cys241, predicting poor binding and low biological
activity. Consequently, we synthesized only three 2-carbon
bridge analogues (21-23) to test the model, and the experi-
mental data confirmed the predictions of the docking results
(Table 1 data).
Replacement of the indole ring with differently substituted
pyrrole and imidazole analogues led to less clear results.
Most of the docked structures showed the trimethoxyphenyl
group in a position similar to that of the corresponding ring
A of the cocrystallized DAMA-colchicine. However, the
heterocycle moieties were placed in different positions com-
pared with the indole ring of the ATIs, and none of the poses
obtained showed any interaction with Thr179 (Figure 4).
From these results, it was not easy to predict the potential
activity of such compounds, and therefore a small series of
pyrrole and imidazole analogues was prepared and tested
experimentally. The biological data clearly showed that, in
our model, the correct position of the trimethoxyphenyl
group is necessary, but not sufficient, to predict the activity
of novel compounds and that other structural features are
required to have an accurate model. It should also be noted
that our findings are in accord with the pharmacophore
model previously developed for colchicine site compounds.21
Conclusions
Replacement of the sulfur atom of arylthioindoles with a
carbonyl functionality led to compounds endowed with com-
parable inhibition of tubulin assembly. Replacement of the
carbonyl with a methylene moiety led to inhibitors of tubulin
assembly whose potency was dependent on the substituent at
position 2 of the indole ring. As growth inhibitors of MCF-7
human breast carcinoma cells, sulfur derivativeswere superior
or equivalent to the ketones, while methylene derivatives were
generally less effective. Compounds bearing either a bromine
atom or a methoxy group at position 5 and the 2-methox-
ycarbonyl group at position 2 of the indole ring were potent
inhibitors of both tubulin polymerization and MCF-7 cell
growth, with potencies comparable to those of reference
compound 2. Two methylene derivatives, 29 and 41, were
also highly active in both assays. Esters 24, 27-29, 36, 39, and
41 showed ∼50% inhibition of human HeLa and HCT116/
chr3 cell growth at 0.5 μM, and these compounds also reduced
cell viability of HEK, M14, and U937 cells, with IC50’s in the
78-220 nM range. In contrast, murine macrophage J744.1
cell growth was significantly less affected (20% at the highest
concentrations). Four additional nontransformed cell lines,
however, did not greatly differ in their sensitivity to com-
pounds 24, 27-29, 36, 39, and 41. The effect of drug treatment
on cell morphology was examined by time-lapse microscopy.
After 48 h, HEK and M14 cells treated with 24, 27-29, 36, 39,
Figure 2. Effect of compounds 24, 27-29, and 54 on BY4741 wild
type strain. Growth was recorded after 3 days at 28 ꢀC; final
compound concentration was 20 μM. a=serial cell dilution (SCD).
Figure 3. Docking poses for compounds 36, 39, and 41 (DAMA-colchicine in green).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7521
Figure 4. Docking poses for compounds 54 and 56 (compound 36 in green).
or 41 showed significant morphologic changes (cells became
rounded with large vacuoles and with a tendency to detach
from the substrate). In a protocol set up to evaluate
compound toxicity on S. cerevisiae BY4741 wild type
strain, compounds 24 and 54 strongly reduced cell growth,
and 29, 36, and 39 also showed significant inhibitory effects.
The strong activity of 54, which was inactive against bovine
tubulin and MCF-7 cells against BY4741 cells, requires
further investigation.
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 biologically tested compounds
were found within (0.4% of the theoretical values. Purity of tested
compounds was >95%. Compounds 3,12 6,10 9,10 12,12 15,10 18,11
24,12 33,12 36,10 and 4211 were prepared as previously reported.
General Procedure for the Synthesis of Compounds 4, 7, 10, 13,
16, 19, 21, 25, 28, 31, 34, 39, and 43. Example: (1H-Indol-3-
yl)-(3,4,5-trimethoxyphenyl)methanone (4). A mixture of AlCl3
(0.57 g; 0.0043 mol), the appropriate 1H-indole (0.5 g, 0.0043
mol) and 3,4,5-trimethoxybenzoyl chloride (0.99 g, 0.0043 mol)
in 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 while
stirring. Once 110 ꢀC was reached, taking about 1 min, the
reaction mixture was held at this temperature for 2 min, then
cooled, diluted cautiously with water, and extracted with
chloroform; the organic layer was washed with brine, dried,
and filtered. Removal of the solvent gave a residue that was
purified by silica gel column chromatography (ethyl acetate:n-
hexane=1:1 as eluent) to furnish 4 as a white solid (0.71 g, 53%),
mp 210-213 ꢀC (from ethanol), differing from lit.22 174-176 ꢀC
and lit.23 132-133 ꢀC. 1H NMR (DMSO-d6): δ 3.73 (s, 3H), 3.83
(s, 6H), 7.06 (s, 2H), 7.20-7.24 (m, 2H), 7.48 (d, J=9.1 Hz, 1H),
8.06 (d, J=3.0 Hz, 1H), 8.21 (d, J=8.5 Hz, 1H), 11.99 ppm
(broad s, disappeared on treatment with D2O, 1H). IR: ν 1571,
Experimental Section
Chemistry. MW-assisted reactions were performed on Dis-
cover LabMate (CEM), setting temperature, irradiation power,
maximum pressure (Pmax), PowerMAX (in situ cooling during
the MW irradiation), ramp and hold times, and open and closed
vessel modes as indicated. Melting points (mp) were determined
on a Buchi 510 apparatus and are uncorrected. Infrared spectra
(IR) were run on a SpectrumOne FT-ATR spectrophotometer.
Band position and absorption ranges are given in cm-1. Proton
nuclear magnetic resonance (1H NMR) spectra were recorded
on Bruker 200 and 400 MHz FT spectrometers in the indicated
solvent. Chemical shifts are expressed in δ units (ppm) from
tetramethylsilane. 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 TLC cards from Fluka (aluminum oxide precoated alu-
minum cards with fluorescent indicator visualizable at 254 nm)
and silica gel TLC cards from Macherey-Nagel (silica gel pre-
coated aluminum cards with fluorescent indicator visualizable
at 254 nm) were used for thin layer chromatography (TLC).
Developed plates were visualized by a Spectroline ENF 260C/F
UV apparatus. Organic solutions were dried over anhydrous
sodium sulfate. Evaporation of the solvents was carried out on a
3179 cm-1
.
Methyl 3-(3,4,5-trimethoxybenzoyl)-1H-indole-2-carboxylate
(7). Synthesized as 4, starting from methyl 1H-indole-2-carbox-
ylate, yield 55%, yellow solid, mp 163-168 ꢀC (from ethanol/
water). 1H NMR (DMSO-d6): δ 3.52 (s, 3H), 3.73 (s, 6H), 3.74 (s,
3H), 7.03 (s, 2H), 7.17 (t, J = 7.2 Hz, 1H), 7.34 (t, J=7.2 Hz,
1H), 7.54 (d, J = 7.5 Hz, 1H), 7.60 (d, J=7.3 Hz, 1H), 12.56 ppm
(broad s, disappeared on treatment with D2O, 1H). IR: ν 1634,
1710, 3279 cm-1. Anal. (C20H19NO6 (369.37)) C, H, N.

7522 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
La Regina et al.
Ethyl 3-(3,4,5-Trimethoxybenzoyl)-1H-indole-2-carboxylate (10).
Synthesized as 4, starting from ethyl 5-methoxy-1H-indole-2-
carboxylate, yield 39%, white solid, mp 105-110 ꢀC (ethanol/n-
hexane). 1H NMR (CDCl3): δ 1.01 (t, J=7.1 Hz, 3H), 3.82 (s,
6H), 3.93 (s, 3H), 4.14 (q, J=7.1 Hz, 2H), 7.18 (s, 2H), 7.24 (t,
J=7.1 Hz, 1H), 7.41 (t, J=7.1 Hz, 1H), 7.5 (d, J=8.3 Hz, 1H),
7.73 (d, J = 8.1 Hz, 1H), 9.26 ppm (broad s, disappeared on
treatment with D2O, 1H). IR: ν 1648, 1686, 3303 cm-1. Anal.
(C21H21NO6 (383.39)) C, H, N.
(5-Methoxy-1H-indol-3-yl)-(3,4,5-trimethoxyphenyl)methanone
(34). Synthesized as 4, starting from 5-methoxy-1H-indole, yield
42%, yellow solid, mp 202-207 ꢀC (from ethanol), lit.14 194-
195 ꢀC.
Methyl 5-Methoxy-3-(3,4,5-trimethoxybenzoyl)-1H-indole-2-
carboxylate (39). Synthesized as 4, starting from methyl 5-
methoxy-1H-indole-2-carboxylate, yield 20%, yellow solid,
1
mp 168-173 ꢀC (from ethanol/water). H NMR (DMSO-d6):
δ 3.47 (s, 3H), 3.71 (s, 3H), 3.73 (s, 3H), 3.74 (s, 6H), 6.99-
7.02 (m, 3H), 7.07 (d, J=2.3 Hz, 1H), 7.44 (d, J=8.95 Hz, 1H),
12.49 ppm (broad s, disappeared on treatment with D2O, 1H).
IR: ν 1635, 1710, 3274 cm-1. Anal. (C21H21NO7 (399.39))
C, H, N.
(5-Chloro-1H-indol-3-yl)-(3,4,5-trimethoxyphenyl)methanone
(13). Synthesized as 4, starting from 5-chloro-1H-indole, yield
1
68%, white solid, mp 240-245 ꢀC (from ethanol). H NMR
(DMSO-d6): δ 3.76 (s, 3H), 3.86 (s, 6H), 7.09 (s, 2H), 7.28 (dd,
J=8.7 Hz, 1H), 7.54 (t, J=8.1 Hz, 1H), 8.19 (s, 1H), 8.23 (d,
J=2.2 Hz, 1H), 12.21 ppm (broad s, disappeared on treatment
with D2O, 1H). IR: ν 1600, 3261 cm-1. Anal. (C18H16ClNO4
(345.78)) C, H, Cl, N.
Ethyl 5-Methoxy-3-(3,4,5-trimethoxybenzoyl)-1H-indole-2-car-
boxylate (43). Synthesized as 4, starting from ethyl 5-methoxy-
1H-indole-2-carboxylate, yield 60%, yellow solid, mp 150-155
ꢀC (from ethanol). 1H NMR (CDCl3): δ 0.97 (t, J=7.1 Hz, 3H),
3.82 (s, 3H), 3.84 (s, 6H), 3.94 (s, 3H), 4.09 (q, J=7.1 Hz, 2H),
7.07 (dd, J=6.5 Hz, 1H), 7.18 (s, 2H), 7.26 (d, J=1.9 Hz, 1H),
7.39 (d, J = 9.0 Hz, 1H), 9.21 ppm (broad s, disappeared on
treatment with D2O, 1H). IR: ν 1647, 1694, 3353 cm-1. Anal.
(C22H23NO7 (413.42)) C, H, N.
General Procedure for the Synthesis of Compounds 5, 14, 26,
and 35. Example: 3-(3,4,5-trimethoxybenzyl)-1H-indole (5). So-
dium borohydride (0.80 g, 0.021 mol) was added to a solution of
4 (0.66 g, 0.0021 mol) in ethanol (85 mL). The reaction mixture
was refluxed for 3 h, then cooled, cautiously diluted with water,
and extracted with ethyl acetate; the organic layer was washed
with brine, dried, and filtered. Evaporation of the solvent gave a
residue that was purified by silica gel column chromatography
(ethyl acetate:n-hexane=1:1 as eluent) to furnish 5 as a white
solid (0.26 g, 42%), mp 133-136 ꢀC (from ethanol), lit.22
125-126 ꢀC and lit.24 128-130 ꢀC.
Methyl 5-Chloro-3-(3,4,5-trimethoxybenzoyl)-1H-indole-2-car-
boxylate (16). Synthesized as 4, starting from methyl 5-chloro-
1H-indole-2-carboxylate, yield 68%, yellow solid, mp 188-
193 ꢀC (from ethanol/water). 1H NMR (CDCl3): δ 3.69 (s, 3H),
3.84 (s, 6H), 3.96 (s, 3H), 7.16 (s, 2H), 7.36 (dd, J = 8.8 Hz,
1H), 7.44 (t, J = 8.8 Hz, 1H), 7.66 (d, J = 1.9 Hz, 1H), 9.50
ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν
1645, 1711, 3259 cm-1. Anal. (C20H18ClNO6 (403.81)) C, H,
Cl, N.
Ethyl 5-Chloro-3-(3,4,5-trimethoxybenzoyl)-1H-indole-2-car-
boxylate (19). Synthesized as 4, starting from ethyl 5-chloro-1H-
indole-2-carboxylate, yield 38%, yellow solid, mp 160-163 ꢀC
(from ethanol). 1H NMR (DMSO-d6): δ 0.86 (t, J=7.1 Hz, 3H),
3.73 (s, 3H), 3.75 (s, 6H), 3.96 (q, J=7.1 Hz, 2H), 7.04 (s, 2H),
7.38 (dd, J= 10.1 Hz, 1H), 7.58 (d, J=7.4 Hz 1H), 7.69 (d,
J = 2.1 Hz, 1H), 12.78 ppm (broad s, disappeared on treat-
ment with D2O, 1H). IR: ν 1636, 1684, 3294 cm-1. Anal.
(C21H20ClNO6 (417.84)) C, H, Cl, N.
Ethyl 5-Chloro-3-(2-(3,4,5-trimethoxyphenyl)acetyl)-1H-in-
dole-2-carboxylate (21). Synthesized as 4, starting from ethyl
5-chloro-1H-indole-2-carboxylate and 2-(3,4,5-trimethoxyphe-
nyl)acetyl chloride, yield 32%, yellow solid, mp 129-134 ꢀC
(from ethanol). 1H NMR (CDCl3): δ 1.46 (t, J = 7.1 Hz, 3H),
3.80 (s, 6H), 3.83 (s, 3H), 4.40 (s, 2H), 4.50 (q, J = 7.1 Hz, 2H),
6.46 (s, 2H), 7.32 (dd, J = 8.8 Hz, 1H), 7.33 (dd, J = 9.5 Hz, 1H),
7.96 (d, J = 1.8 Hz, 1H), 9.22 ppm (broad s, disappeared on
treatment with D2O, 1H). IR: ν 1641, 1721, 3169 cm-1. Anal.
(C22H22ClNO6 (431.87)) C, H, Cl, N.
5-Chloro-3-(3,4,5-trimethoxybenzyl)-1H-indole (14). Synthe-
sized as 5 starting from 13, yield 47%, orange solid, mp 145-
150 ꢀC (from ethanol). 1H NMR (CDCl3): δ 3.80 (s, 6H), 3.83 (s,
3H), 4.00 (s, 2H), 6.49 (s, 2H), 6.95 (d, J=2.3 Hz, 1H), 7.14 (dd,
J=8.6 Hz, 1H), 7.28 (d, J=8.1 Hz, 1H), 7.51 (d, J=2.0 Hz, 1H),
8.40 ppm (broad s, disappeared on treatment with D2O, 1H). IR:
ν 3372 cm-1. Anal. (C18H18ClNO3 (331.79)) C, H, Cl, N.
5-Bromo-3-(3,4,5-trimethoxybenzyl)-1H-indole (26). Synthe-
sized as 5, starting from 25, yield 58%, white solid, mp 145-
150 ꢀC (from ethanol). 1H NMR (CDCl3): 3.81 (s, 6H), 3.84 (s,
3H), 4.01 (s, 2H), 6.50 (s, 2H), 6.94 (d, J = 2.3 Hz, 1H), 7.24-
7.28 (m, 2H), 7.69 (d, J = 1.7 Hz, 1H), 8.05 ppm (broad s,
disappeared on treatment with D2O, 1H). IR: ν 3353 cm-1
.
(5-Bromo-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)methanone
(25). Synthesized as 4, starting from 5-bromo-1H-indole, yield
Anal. (C18H18BrNO3)) C, H, Br, N.
1
5-Methoxy-3-(3,4,5-trimethoxybenzyl)-1H-indole (35). Synt-
hesized as 5, starting from 34, yield 68%, white solid, mp 110-
113 ꢀC (from ethanol). 1H NMR (CDCl3): δ 3.78 (s, 6H), 3.80 (s,
3H), 3.81 (s, 3H), 4.01 (s, 2H), 6.51 (s, 2H), 6.84 (d, J=8.8 Hz,
1H), 6.88 (t, J=2.4 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 7.24 (t,
J=4.7 Hz, 1H), 7.90 ppm (broad s, disappeared on treatment
with D2O, 1H). IR: ν 3365 cm-1. Anal. (C19H21NO4 (327.37)) C,
H, N.
General Procedure for the Synthesis of Compounds 8, 11, 17,
20, 23, 29, 32, 41, and 45. Example: Methyl 3-(3,4,5-
trimethoxybenzyl)-1H-indole-2-carboxylate (8). To a cold solu-
tion of 7 (0.31 g, 0.00084 mol) in trifluoroacetic acid (0.96 g,
0.65 mL, 0.0084 mol) was added dropwise triethylsilane (0.22 g,
0.30 mL, 0.0019 mol). The reaction mixture was stirred at 25 ꢀC
for 24 h, then neutralized with a saturated solution of sodium
hydrogen carbonate and extracted with ethyl acetate; the or-
ganic layer was washed with brine, dried, and filtered. Removal
of the solvent gave a residue that was purified by silica gel
column chromatography (ethyl acetate:n-hexane=3:2 as elu-
ent) to furnish 8 as a yellow solid (0.15 g, 50%), mp 156-161 ꢀC
(from ethanol/n-hexane). 1H NMR (CDCl3): δ 3.69 (s, 6H), 3.72
(s, 3H), 3.89 (s, 3H), 4.38 (s, 2H), 6.46 (s, 2H), 7.05 (t, J = 8.1 Hz,
1H), 7.25 (t, J = 8.3 Hz, 1H), 7.33 (d, J = 7.4 Hz, 1H), 7.57
53%, yellow solid, mp 235-240 ꢀC (from ethanol). H NMR
(DMSO-d6): δ 3.76 (s, 3H), 3.86 (s, 6H), 7.09 (s, 2H), 7.40 (dd,
J=6.6 Hz, 1H), 7.50 (d, J=8.1 Hz, 1H), 8.18 (s, 1H), 8.39 (d,
J=2.5 Hz, 1H), 12.19 ppm (broad s, disappeared on treatment
with D2O, 1H). IR: ν 1599, 3279 cm-1. Anal. (C18H16BrNO4
(390.23)) C, H, Br, N.
Methyl 5-Bromo-3-(3,4,5-trimethoxybenzoyl)-1H-indole-2-
carboxylate (28). Synthesized as 4, starting from 49, yield
40%, white solid, mp 207-210 ꢀC (from ethanol/n-hexane).
1H NMR (CDCl3): δ 3.68 (s, 3H), 3.83 (s, 6H), 3.95 (s, 3H), 7.14
(s, 2H), 7.38 (d, J=8.2 Hz, 1H), 7.48 (dd, J = 6.9 Hz, 1H), 7.81
(d, J=1.8 Hz, 1H), 9.45 ppm (broad s, disappeared on treat-
ment with D2O, 1H). IR: ν 1642, 1711, 3248 cm-1. Anal.
(C20H18BrNO6 (448.26)) C, H, Br, N.
Ethyl 5-Bromo-3-(3,4,5-trimethoxybenzoyl)-1H-indole-2-car-
boxylate (31). Synthesized as 4, starting from 48, yield 46%,
yellow solid, mp 135-140 ꢀC (from ethanol/n-hexane). 1H
NMR (DMSO-d6): δ 0.85 (t, J = 7.1 Hz, 3H), 3.73 (s, 3H),
3.75 (s, 6H), 3.98 (q, J=7.1 Hz, 2H), 7.03 (s, 2H), 7.33 (d, J=1.2
Hz, 1H), 7.48 (dd, J = 6.9 Hz, 1H), 7.53 (dd, J = 8.8 Hz,
1H), 12.74 ppm (broad s, disappeared on treatment with
D2O, 1H). IR: ν 1638, 1702, 3292 cm-1. Anal. (C21H20BrNO6
(462.29)) C, H, Br, N.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7523
(d, J=8.2 Hz, 1H), 8.75 ppm (broad s, disappeared on treatment
with D2O, 1H). IR: ν 1690, 3318 cm-1. Anal. (C20H21NO5
(355.38)) C, H, N.
5-methoxy-1H-indole-2-carboxylate (40). A mixture of 39 (0.34
g, 0.00085 mol) and sodium borohydride (0.03 g, 0.00085 mol) in
THF (2.1 mL) and water (0.12 mL) was refluxed for 2 h. After
cooling, water and ethyl acetate were added. The organic layer
was removed and washed with brine, dried, and filtered. Re-
moval of the solvent gave a residue that was purified by silica gel
column chromatography (ethyl acetate:n-hexane=1:1 as eluent)
to furnish 40 as a brown solid (0.1 g, 29%), mp 100-103 ꢀC
(from ethanol). 1H NMR (CDCl3): δ 3.76 (s, 3H), 3.79 (s, 6H),
3.82 (s, 3H), 3.97 (s, 3H), 4.53 (d, J = 1.2 Hz, 1H), 6.50 (d,
J=7.4 Hz, 1H), 6.77 (s, 2H), 6.95 (d, J=2.4 Hz, 1H), 7.02 (dd,
J=6.5 Hz, 1H), 7.28 (d, J=8.4 Hz, 1H), 8.78 ppm (broad s,
disappeared on treatment with D2O, 1H). IR: ν 1707, 2937,
3322 cm-1. Anal. (C21H23NO7 (401.41)) C, H, N.
Ethyl 3-(Hydroxy(3,4,5-trimethoxyphenyl)methyl)-5-methoxy-
1H-indole-2-carboxylate (44). Synthesized as 40, starting from 43,
yield 53%, white solid, mp 115-120 ꢀC (from ethanol). 1H NMR
(CDCl3): δ 1.42 (t, J = 7.2 Hz, 3H), 3.76 (s, 3H), 3.79 (s, 3H), 3.81
(s, 3H), 3.83 (s, 3H), 4.43 (q, J = 6.0 Hz, 2H), 4.44 (d, J = 1.2 Hz,
1H), 6.49 (d, J = 7.2 Hz, 1H), 6.77 (s, 2H), 6.95 (d, J = 2.3 Hz,
1H), 7.01(dd, J = 6.6 Hz, 1H), 7.29(d, J = 7.3Hz, 1H), 8.78 ppm
(broad s, disappeared on treatment with D2O, 1H). IR: ν 1676,
3209, 3402 cm-1. Anal. (C22H25NO7 (415.44)) C, H, N.
Ethyl 3-(3,4,5-Trimethoxybenzyl)-1H-indole-2-carboxylate
(11). Synthesized as 8, starting from 10, yield 73%, white solid,
1
mp 115-118 ꢀC (from ethanol/n-hexane). H NMR (CDCl3):
δ 1.42 (t, J=7.1 Hz, 3H), 3.77 (s, 6H), 3.80 (s, 3H), 4.44 (q, J=
7.1 Hz, 2H), 4.47 (s, 2H), 6.55 (s, 2H), 7.13 (t, J=7.0 Hz, 1H),
7.33 (t, J=6.9 Hz, 1H), 7.39 (d, J=7.5 Hz, 1H), 7.64 (d, J=7.4
Hz, 1H), 8.82 ppm (broad s, disappeared on treatment with
D2O, 1H). IR: ν 1672, 3337 cm-1. Anal. (C21H23NO5) (369.41))
C, H, N.
Methyl 5-Chloro-3-(3,4,5-trimethoxybenzyl)-1H-indole-2-car-
boxylate (17). Synthesized as 8, starting from 16, yield 69%,
white solid, mp 172-175 ꢀC (from ethanol/n-hexane). 1H NMR
(CDCl3): δ 3.70 (s, 6H), 3.73 (s, 3H), 3.89 (s, 3H), 4.33 (s, 2H),
6.42 (s, 2H), 7.21 (d, J=1.9 Hz, 1H), 7.24 (t, J=8.7 Hz, 1H), 7.51
(d, J=1.9 Hz, 1H), 8.74 ppm (broad s, disappeared on treatment
with D2O, 1H). IR: ν 1682, 3322 cm-1. Anal. (C20H20ClNO5
(389.83)) C, H, Cl, N.
Ethyl 5-Chloro-3-(3,4,5-trimethoxybenzyl)-1H-indole-2-carbo-
xylate (20). Synthesized as 8, starting from 19, yield 95%, yellow
solid, mp 147-150 ꢀC (from ethanol/n-hexane). 1H NMR
(CDCl3): δ 1.41 (t, J=7.1 Hz, 3H), 3.79 (s, 6H), 3.81 (s, 3H),
4.41 (s, 2H), 4.43 (q, J=6.4 Hz, 2H), 6.51 (s, 2H), 7.25-7.28 (m,
1H), 7.33 (dd, J=8.1 Hz, 1H), 7.59-7.60 (m, 1H), 8.91 ppm
(broad s, disappeared on treatment with D2O, 1H). IR: ν 1666,
3315 cm-1. Anal. (C21H22ClNO5)) C, H, Cl, N.
Ethyl 5-Chloro-3-(2-oxo-2-(3,4,5-trimethoxyphenyl)acetyl)-1H-
indole-2-carboxylate (22). A mixture of 21 (0.05 g, 0.00012 mol)
and selenium(IV) oxide (0.05 g; 0.00045 mol) in DMSO (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 150 ꢀC. Once 150 ꢀC was reached, taking about
1 min, the reaction mixture was held at this temperature for 2 min,
while stirring, then cooled, diluted with water, and extracted with
ethyl acetate; organic layer was washed with brine, dried, and
filtered. Removal of the solvent gave a residue that was purified by
silica gel column chromatography (ethyl acetate:n-hexane=1:2 as
eluent) to furnish 22 as a yellow solid (0.03 g, 56%), mp 177-
181 ꢀC (from ethanol). 1H NMR (CDCl3): δ 1.07(t, J=7.2 Hz,
3H), 3.93(s,6H), 3.98(s,3H), 4.12 (q, J=7.1 Hz, 2H), 7.35 (s, 2H),
7.42-7.43 (m, 2H), 8.40 (s, 1H), 9.50 ppm (broad s, disappeared
Ethyl 5-Chloro-3-(3,4,5-trimethoxyphenethyl)-1H-indole-2-carbo-
xylate (23). Synthesized as 8, starting from 22, yield 40%, white
solid, mp 163-165 ꢀC (from ethanol). 1H NMR (CDCl3): δ 1.44 (t,
J=7.5 Hz, 3H), 2.88 (t, J=7.4 Hz, 2H), 3.35 (t, J=7.3 Hz, 2H), 3.79
(s, 6H), 3.83 (s, 3H), 4.42 (t, J=7.1 Hz, 2H), 6.38 (s, 2H), 7.24 (dd,
J=9.2 Hz, 1H), 7.31 (d, J=8.8 Hz, 1H), 7.49 (d, J=1.9 Hz, 1H),
9.01 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν
1669, 3318 cm-1. Anal. (C22H24ClNO5 (417.88)) C, H, Cl, N.
Methyl 5-Bromo-3-(3,4,5-trimethoxybenzyl)-1H-indole-2-car-
boxylate (29). Synthesized as 8, starting from 28, yield 97%,
yellow solid, mp 177-182 ꢀC (from ethanol/n-hexane). 1H
NMR (DMSO-d6): δ 3.57 (s, 3H), 3.67 (s, 6H), 3.92 (s, 3H),
4.34 (s, 2H), 6.62 (s, 2H), 7.36-7.36 (m, 2H), 7.90 (d, J=2.3 Hz,
1H), 11.88 ppm (broad s, disappeared on treatment with D2O,
1H). IR: ν 1675, 3322 cm-1. Anal. (C20H20BrNO5 (434.28)) C,
H, Br, N.
Ethyl 5-Bromo-3-(3,4,5-trimethoxybenzyl)-1H-indole-2-car-
boxylate (32). Synthesized as 8, starting from 31, yield 46%,
white solid, mp 150-155 ꢀC (from ethanol/n-hexane). 1H NMR
(CDCl3): δ 1.41 (t, J=7.1 Hz, 3H), 3.79 (s, 6H), 3.82 (s, 3H), 4.43
(q, J=7.1 Hz, 2H), 6.51 (s, 2H), 7.28 (d, J=9.4 Hz, 1H), 7.29 (s,
2H), 7.40 (dd, J = 6.9 Hz, 1H), 7.77 (d, J = 1.8 Hz, 1H), 8.90
ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν
1665, 3317 cm-1. Anal. (C21H22BrNO6 (448.31)) C, H, Br, N.
Methyl 5-Methoxy-3-(3,4,5-trimethoxybenzyl)-1H-indole-2-
carboxylate (41). Synthesized as 8, starting from 39, yield
58%, white solid, mp 125-128 ꢀC (from ethanol/n-hexane).
1H NMR (CDCl3): δ 3.77 (s, 6H), 3.79 (s, 3H), 3.80 (s, 3H), 3.94
(s, 3H), 4.42 (s, 2H), 6.53 (s, 1H), 6.96 (d, J=2.3 Hz, 1H), 7.00
(dd, J=6.4 Hz, 1H), 7.28 (d, J=8.9 Hz, 1H), 8.67 (s, 1H), 11.91
ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν
1687, 3331 cm-1. Anal. (C21H23NO6 (385.41)) C, H, N.
Ethyl 5-Methoxy-3-(3,4,5-trimethoxybenzyl)-1H-indole-2-car-
boxylate (45). Synthesized as 8, starting from 43, yield 90%,
white solid, mp 132-135 ꢀC (from ethanol). 1H NMR (CDCl3):
δ 1.42 (t, J=7.1 Hz, 3H), 3.78 (s, 9H), 3.81 (s, 3H), 4.44 (q, J=
7.2 Hz, 2H), 4.45 (s, 2H), 6.56 (s, 2H), 6.98-7.03 (m, 2H), 7.28-
7.32 (m, 1H), 8.76 ppm (broad s, disappeared on treatment with
D2O, 1H). IR: ν 1672, 3330 cm-1. Anal. (C22H25NO6 (399.44))
C, H, N.
on treatment with D2O, 1H). IR: ν 1650, 1672, 1724, 3275 cm-1
.
Anal. (C22H20ClNO7 (445.85)).
Methyl 5-Bromo-3-(3,4,5-trimethoxyphenylthio)-1H-indole-2-
carboxylate (27). A mixture of 59 (0.25 g, 0.001 mol), 3,4,5-
bis(3,4,5-trimethoxyphenyl)disulfide10 (0.55 g, 0.0014 mol), and
sodium hydride (0.071 g, 0.003 mol, 60% in mineral oil) in
anhydrous DMF (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. Once 110 ꢀC was
reached, taking about 1 min, the reaction mixture was held at this
temperature for 2 min, while stirring, then cooled and quenched
on crushed ice and extracted with ethyl acetate. The organic layer
was washed with brine, dried, and filtered. Removal of the solvent
gave crude 5-bromo-3-(3,4,5-trimethoxyphenylthio)-1H-indole-
2-carboxylic acid, which was used without further purification.
The crude acid was dissolved in methanol (2.5 mL) and dichlor-
omethane (10 mL) and treated with TMSDM (0.78 mL, 0.0015
mol, 2.0 M in hexane) while stirring at 25 ꢀC for 30 min. Removal
of the solvent gave a crude product that was purified by silica gel
column chromatography (ethyl acetate:n-hexane=1:1) to furnish
27 as a white solid (0.36 g, overall yield 79%), mp 160-162 ꢀC
1
(from ethanol). H NMR (CDCl3): δ 3.72 (s, 6H), 3.81 (s, 3H),
3.98 (s, 3H), 6.48 (s, 2H), 7.33 (d, J=9.3 Hz, 1H), 7.42 (dd, J=1.9
and 9.3 Hz, 1H), 7.72 (d, J=1.9 Hz, 1H), 9.20 ppm (broad s,
disappeared on treatment with D2O, 1H). IR: ν 1680, 3296 cm-1
Anal. (C19H18BrNO5S (452.32))
.
Ethyl 5-Bromo-3-(3,4,5-trimethoxyphenylthio)-1H-indole-2-car-
boxylate (30). Synthesized as 27, starting from 59 and 3,4,5-
bis(3,4,5-trimethoxyphenyl)disulfide.10 The crude acid (0.5 g)
was dissolved in absolute ethanol (1.7 mL) and treated with
thionyl chloride (0.13 mL) at 0 ꢀC under an Ar stream. The
reaction mixture was stirred at 0 ꢀC for 20 min, heated at 65 ꢀC for
General Procedure for the Synthesis of Compounds 40 and 44.
Example: Methyl 3-(Hydroxy(3,4,5-trimethoxyphenyl)methyl)-

7524 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
La Regina et al.
2 h, then cooled and diluted with water and ethyl acetate. The
organic layer was removed and washed with a saturated solution
of sodium hydrogen carbonate and brine, dried, and filtered.
Removal of the solvent gave a crude product that was purified
by silica gel column chromatography (ethyl acetate:n-hexane =
1:1) to give 30 as a yellow solid (0.33 g, overall yield 70%), mp
4.15 (q, J=7.1 Hz, 2H), 5.44 (s, 2H), 5.96 ppm (s, 2H). IR: ν
1708 cm-1. Anal. (C22H31NO5 (389.49)) C, H, N.
Ethyl 1-(3,4,5-Trimethoxybenzyl)-1H-imidazole-4-carboxylate
(50). A solution of DIPAD (1.29 g, 1.25 mL, 0.0064 mol) in
anhydrous THF (18 mL) was added dropwise to a mixture of
3,4,5-trimethoxybenzyl alcohol (1.26 g, 0.0064 mol), ethyl 1-H-
imidazole-4-carboxylate26 (1.0 g, 0.0071 mol), and anhydrous
triphenylphosphine (1.86 g, 0.0071 mol) in the same solvent
(35 mL). The reaction was stirred at 25 ꢀC overnight under an
Ar stream. The reaction mixture was concentranted in vacuo,
diluted with water, and extracted with ethyl acetate. The organic
layer was washed with brine, dried, and filtered. Removal of the
solvent gave a residue that was purified by silica gel column
chromatography (ethyl acetate:methanol = 45:5 as eluent) to
furnish 50 as a colorless oil (0.2 g, 10%). 1H NMR (CDCl3): δ
1.26 (t, J=7.4 Hz, 3H), 3.72 (s, 6H), 3.75 (s, 3H), 4.22 (q, J=
7.1 Hz, 2H), 5.36 (s, 2H), 6.33 (s, 2H), 7.54 (s, 1H), 7.69 ppm (s,
1H). IR: ν 1695 cm-1. Anal. (C16H20N2O5 (320.34)) C, H, N.
Ethyl 1-(3,4,5-Trimethoxybenzoyl)-1H-pyrrole-2-carboxylate
(51). To a stirred mixture of potassium tert-butoxide (0.26 g,
0.0023 mol) and 18-crown-6 (0.47 g, 0.0018 mol) in anhydrous
THF (25 mL) was added dropwise a solution of ethyl 1H-
pyrrole-2-carboxylate (0.25 g, 0.0018 mol) in the same solvent
(25 mL). After cooling to 0 ꢀC for 15 min, a solution of 3,4,5-
trimethoxybenzoyl chloride (0.41 g, 0.0018 mol) in the same
solvent (25 mL) was added dropwise. The reaction mixture was
stirred at 25 ꢀC for 4 h and then concentranted to a small volume
and extracted with ethyl acetate. The organic layer was washed
with brine, dried, and filtered. Removal of the solvent gave a
residue that was purified by silica gel column chromatography
(dichloromethane as eluent) to furnish 51 as an orange oil (0.05
g, 8%). 1H NMR (CDCl3): δ 1.09 (t, J=7.1 Hz, 3H), 3.78 (s, 6H),
3.82 (s, 3H), 4.02 (q, J=7.1 Hz, 2H), 6.24 (m, 1H), 6.94 (s, 2H),
6.99 (m, 1H), 7.18 ppm (m, 1H). IR: ν 1670, 1703 cm-1. Anal.
(C17H19NO6 (333.34)) C, H, N.
1
150-152 ꢀC (from ethanol). H NMR (CDCl3): δ 1.37 (t, J=
7.1 Hz, 3H), 3.72 (s, 6H), 3.80 (s, 3H), 4.43 (q, J=7.8 Hz, 2H), 6.48
(s, 2H), 7.33 (d, J =8.7 Hz, 1H), 7.43-7.46 (m, 1H) 7.74-7.75 (m,
1H), 9.23 ppm (broad s, disappeared on treatment with D2O, 1H).
IR: ν 1672, 3299 cm-1. Anal. (C20H20BrNO5S (452.32)).
Methyl 5-Methoxy-3-(3,4,5-trimethoxyphenylsulfinyl)-1H-
indole-2-carboxylate (37). To a cold solution of 3610 (0.05 g,
0.00012 mol) in chloroform (5 mL) was added 3-chloroperoxy-
benzoic acid (0.021 g, 0.00012 mol). The reaction mixture was
stirred at 25 ꢀC for 1.5 h, then diluted with water and extracted
with chloroform. The organic layer was washed with brine,
dried, and filtered. Removal of the solvent gave a residue that
was purified by silica gel column chromatography (ethyl acetate
as eluent) to furnish 37 as a brown solid (0.05 g, yield 99%), mp
161-163 ꢀC (from ethanol). 1H NMR (CDCl3): δ 3.74 (s, 3H),
3.84 (s, 9H), 3.85 (s, 3H), 6.96 (d, J=9.1 Hz, 1H), 7.10 (s, 2H),
7.31 (dd, J=1.9 and 9.0 Hz, 1H), 7.42 (d, J=2.2 Hz, 1H), 9.37
ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν
1708, 2831, 2933 cm-1. Anal. (C20H21NO7S (419.45)) C, H, N, S.
Methyl 5-Methoxy-3-(3,4,5-trimethoxyphenylsulfonyl)-1H-
indole-2-carboxylate (38). Was synthesized as 37, treating 3610
with 3-chloroperoxybenzoic acid (2 equiv), 96%, yellow solid,
mp 176-180 ꢀC (from ethanol). 1H NMR (DMSO-d6): δ 3.33 (s,
3H), 3.82 (s, 6H), 3.83 (s, 3H), 3.93 (s, 3H), 7.04 (dd, J=6.5 Hz,
1H), 7.40 (s, 2H), 7.45 (d, J=9.0 Hz, 1H), 7.59 (d, J=2.4 Hz,
1H), 12.99 ppm (broad s, disappeared on treatment with D2O,
1H). IR: ν 1701, 3298 cm-1. Anal. (C20H21NO8S (435.45)) C, H,
N, S.
Ethyl 1-(3,4,5-Trimethoxybenzyl)-1H-pyrrole-2-carboxylate
(46). 3,4,5-Trimethoxybenzyl chloride15 (1.083 g, 0.005 mol) was
added to a well stirred mixture of ethyl 1H-pyrrole-2-carbox-
ylate (0.63 g, 0.0045 mol), tetrabutylammonium hydrogen
sulfate (1.53 g, 0.0045 mol), a 50% potassium hydroxide aqu-
eous solution (15 mL) and dichloromethane (22 mL). The
reaction mixture was stirred at 25 ꢀC overnight, neutralized
with 6 N hydrochloric acid and extracted with chloroform. The
organic layer was washed with brine, dried, and filtered. Re-
moval of the solvent gave a residue that was purified by silica gel
column chromatography (dichloromethane as eluent) to furnish
46 as a yellow oil (0.64 g, 45%). 1H NMR (CDCl3): δ 1.21 (t, J=
7.1 Hz, 3H), 3.68 (s, 6H), 3.71 (s, 3H), 4.15 (q, J=7.1 Hz, 2H),
5.39 (s, 2H), 6.07 (dd, J=3.9 and 2.6 Hz, 1H), 6.24 (s, 2H), 6.78
(t, J=2.1 Hz, 1H), 6.91 ppm (dd, J = 3.9 and 1.8 Hz, 1H). IR: ν
1701 cm-1. Anal. (C17H21NO5 (319.35)) C, H, N.
Ethyl 1-(3,4,5-Trimethoxybenzoyl)-1H-imidazole-4-carboxy-
late (52) and Ethyl 1-(3,4,5-Trimethoxybenzoyl)-1H-imidazole-
5-carboxylate (53). To a solution of ethyl imidazole 4-carbox-
ylate26 (0.5 g, 0.0035 mol) in anhydrous dichloromethane
(10 mL) was added dropwise a solution of 3,4,5-trimethoxyben-
zoyl chloride (0.41 g, 0.0018 mol) in the same solvent (10 mL).
The reaction mixture was refluxed for 2.5 h and, after cooling,
quenched on crushed ice. The organic layer was removed and
washed with brine, dried, and filtered. Removal of the solvent
gave a residue that was purified by silica gel column chroma-
tography (ethyl acetate as eluent). The initial eluate furnished 52
as a white solid (0.17 g, 15%), mp 130-133 ꢀC (from ethanol).
1H NMR (CDCl3): δ 1.34 (t, J=7.1 Hz, 3H), 3.84 (s, 6H), 3.90 (s,
3H), 4.34 (q, J=7.1 Hz, 2H), 6.96 (s, 2H), 8.06 (s, 1H), 8.09 ppm
(s, 1H). IR: ν 1672, 1702 cm-1. Anal. (C16H18N2O6 (334.32)) C,
H, N. Further elution with the same solvent afforded 53 as a
Ethyl 3,5-Dimethyl-1-(3,4,5-trimethoxybenzyl)-1H-pyrrole-2-
carboxylate (47). Was synthesized as 46, starting from ethyl 3,5-
dimethyl-1H-pyrrole-2-carboxylate, yield 56%, pink solid, mp
68-70 ꢀC (from toluene). 1H NMR (CDCl3): δ 1.16 (t, J=7.1
Hz, 3H), 2.02 (s, 3H), 2.22 (s, 3H), 3.61 (s, 6H), 3.66 (s, 3H), 4.10
(q, J=7.1 Hz, 2H), 5.37 (s, 2H), 5.71 (s, 1H), 6.03 ppm (s, 2H).
IR: ν 1703 cm-1. Anal. (C19H25NO5 (347.41)) C, H, N.
1
white solid (0.12 g, 13%), mp 140-143 ꢀC (from ethanol). H
NMR (CDCl3): δ 1.40 (t, J=7.1 Hz, 3H), 3.90 (s, 6H), 3.97
(s, 3H), 4.40 (q, J=7.1 Hz, 2H), 7.02 (s, 2H), 8.16 ppm (s, 2H).
IR: ν 1674, 1707 cm-1. Anal. (C16H18N2O6 (334.32)) C, H, N.
1-(3,4,5-Trimethoxybenzyl)-1H-pyrrole-2-carboxylic acid (54).
A mixture of 46 (0.23 g, 0.00072 mol) and potassium hydroxide
(0.12 g, 0.0022 mol) in methanol:water (1:1) (10 mL) was refluxed
for 3 h. After cooling, the reaction mixture was washed with
dichloromethane; the aqueous layer was made acidic with 2 N
hydrochloric acid (pH = 2) and extracted with dichloromethane.
The organic layer was washed with water, dried, filtered, and
evaporated to give 54 as a brown solid (0.15 g, 74%), mp 80-81
ꢀC (from ethanol). 1H NMR (CDCl3): δ 3.68 (s, 6H), 3.71 (s, 3H),
5.36 (s, 2H), 6.12 (dd, J=3.85 and 2.6 Hz, 1H), 6.27 (s, 2H), 6.85
(t, J=2.0 Hz, 1H), 7.04 (dd, J = 3.9 and 1.7 Hz, 1H), 10.69 ppm
(broad s, disappeared on treatment with D2O, 1H). IR: ν 1703,
2995 cm-1. Anal. (C15H17NO5 (291.30)) C, H, N.
Ethyl 3,4,5-Trimethyl-1-(3,4,5-trimethoxybenzyl)-1H-pyrrole-
2-carboxylate (48). Was synthesized as 46, starting from ethyl
3,4,5-trimethyl-1H-pyrrole-2-carboxylate, yield 45%, yellow
1
solid, mp 82-84 ꢀC (from ethanol). H NMR (CDCl3): δ 0.36
(t, J=7.1 Hz, 3H), 1.03 (s, 3H), 1.17 (s, 3H), 1.36 (s, 3H), 2.81 (s,
6H), 2.86 (s, 3H), 3.30 (q, J=7.1 Hz, 2H), 4.56 (s, 2H), 5.22 ppm
(s, 2H). IR: ν 1710 cm-1. Anal. (C20H27NO5 (361.44)) C, H, N.
Ethyl 3,4-Diethyl-5-methyl-1-(3,4,5-trimethoxybenzyl)-1H-pyr-
role-2-carboxylate(49). Was synthesized as 46, starting from ethyl
3,4-diethyl-5-methyl-1H-pyrrole-2-carboxylate, yield 57%, yel-
1
low oil. H NMR (CDCl3): δ 0.99 (t, J=7.5 Hz, 3H), 1.07 (t,
J=7.3 Hz, 3H), 1.21 (t, J=7.1 Hz, 3H), 2.02 (s, 3H), 2.35 (q, J=
7.5 Hz, 2H), 2.68 (q, J=7.4 Hz, 2H), 3.64 (s, 6H), 3.71 (s, 3H),
2-(2-Hydroxyethoxy)ethyl 3,4,5-Trimethyl-1-(3,4,5-trimetho-
xybenzyl)-1H-pyrrole-2-carboxylate (55). A mixture of 48

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7525
(0.05 g, 0.0014 mol) and potassium hydroxide (0.043 g, 0.00077
mol) in diethylene glycol (1 mL) was heated at 140 ꢀC for 3 h.
After cooling, water and ethyl acetate were added and the layers
separated. The organic layer was washed with brine, dried, and
filtered. Removal of the solvent gave a residue that was purified
by silica gel column chromatography (ethyl acetate as eluent) to
give 55 as a yellow oil (0.02 g, 34%). 1H NMR (CDCl3): δ 1.89 (s,
3H), 2.02 (s, 3H), 2.23 (s, 3H), 3.50 (m, 2H), 3.62 (m, 2H), 3.66
(m, 3H), 3.68 (s, 6H), 3.73 (s, 3H), 4.28 (m, 2H), 5.41 (s, 2H), 6.06
ppm (s, 2H). IR: ν 1705, 2836 cm-1. Anal. (C22H31NO7 (421.28))
C, H, N.
1-(3,4,5-Trimethoxybenzyl)-1H-imidazole-4-carboxylic Acid
Hydrochloride (56). A mixture of 50 (0.08 g, 0.00025 mol) and
37% hydrochloric acid (1.5 mL) was refluxed for 1.5 h. After
cooling, the reaction mixture was washed with ethyl acetate. The
aqueous layer was evaporated in vacuo. The residue was dis-
solved in 2-propanol (1.5 mL) and cooled to -78 ꢀC. Upon
addition of n-hexane, the suspension was filtered to give 56 as a
white solid (0.05 g, 68%), mp 163-166 ꢀC (from methanol). 1H
NMR (CD3OD): δ 3.77 (s, 3H), 3.85 (s, 6H), 5.73 (s, 2H), 6.82 (s,
2H), 8.24 (s, 1H), 9.22 (s, 1H), 12.10 (broad s, disappeared on
treatment with D2O, 1H). IR: ν 1705, 3100 cm-1. Anal.
were separated, and the organic phase was washed with a
saturated solution of sodium hydrogen carbonate and brine,
dried, filtered, and evaporated to give 60 as a white solid (3.43
g, 96%), mp 217-220 ꢀC (from ethanol/cyclohexane), lit.25
211.8-213.6.
2-(3,4,5-Trimethoxyphenyl)acetyl Chloride. This compound
was synthesized by treating a cold solution of 3,4,5-trimethox-
yphenylacetic acid (0.25 g, 0.0011 mol) in anhydrous THF
(5 mL) with oxalyl chloride (0.28 g, 0.19 mL, 0.0022 mol).
Immediately afterward, a catalytic amount (2 drops) of anhy-
drous DMF was added, and vigorous bubbling ensued. After
10 min, the reaction mixture was carefully concentrated in vacuo
to provide a viscous oil. Fresh anhydrous THF was added, and
the solution was concentrated in vacuo again to furnish 2-(3,4,5-
trimethoxyphenyl)acetyl chloride (0.27 g, yellow oil), which was
used without further purification.
Molecular Modeling. All molecular modeling studies were
performed on a MacPro dual 2.66 GHz Xeon running Ubuntu
8. The tubulin structure was downloaded from the PDB (http://
www.rcsb.org/: PDB code 1SA0).3 Hydrogen atoms were added
to the protein, using Molecular Operating Environment (MOE)
2007.09,27 and minimized, keeping all the heavy atoms fixed
until a rmsd gradient of 0.05 kcal mol-1 A-1 was reached. Ligand
structures were built with MOE and minimized using the MM-
FF94x forcefield until a rmsd gradient of 0.05 kcal mol-1 A-1
was reached. The docking simulations were performed using
FlexX28 with the MOE interface. The rmsd of the trimethox-
yphenyl moiety for each of the compounds evaluated was
calculated in comparison with ring A of DAMA-colchicine,
and the results from the docking were scored using this value.29
The images presented here were created with Zodiac.30
Biology. Tubulin Assembly. The reaction mixtures contained
0.8 M monosodium glutamate (pH 6.6 with hydrochloric acid in
2 M stock solution), 10 μM tubulin, and varying concentrations
of drug. 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 con-
trolled recording spectrophotometer for 20 min at 30 ꢀC. Extent
of reaction was measured. Full experimental details were pre-
viously reported.31
(C14H16N2O5 HCl (328.75)) C, H, Cl, N.
3
Ethyl 2-(2-(4-Bromophenyl)hydrazono)propanoate (57). A
mixture of 4-bromophenylhydrazine hydrochloride (5.7 g,
0.0255 mol), ethyl pyruvate (2 g, 1.91 mL, 0.017 mol), and
sodium acetate (2.79 g, 0.034 mol) in ethanol (40 mL) was placed
into the MW cavity (open vessel mode). MW irradiation at 250
W was used, the temperature being ramped from 25 to 100 ꢀC.
Once 100 ꢀC was reached, taking about 2 min, the reaction
mixture was held at this temperature for 5 min while stirring.
The reaction mixture was cooled at 0 ꢀC, with stirring, and
filtered to give 57, yield 90%, mp 142-144 ꢀC (from ethanol). 1H
NMR (CDCl3): δ 1.39 (t, J=7.2 Hz, 3H), 2.12 (s, 3H), 4.33 (q,
J = 7.1 Hz, 2H), 7.10 (d, J=6.7 Hz, 2H), 7.40 (d, J=6.9 Hz, 2H),
7.64 ppm (broad s, disappeared on treatment with D2O, 1H). IR:
ν 1576, 1675, 3290 cm-1. Anal. (C11H13BrN2O2 (285.14)).
Ethyl 5-Bromo-1H-indole-2-carboxylate (58). Compound 57
(35.15 g, 0.123 mol) was added in portions to PPA (350 g)
preheated at 110 ꢀC. The mixture was stirred at the same
temperature for 30 min and then quenched with ice-water.
The solid was filtered, washed with water, dried, and recrystal-
lized from ethanol to give 58 as a brown solid (32.95 g, 60%), mp
160-161 ꢀC. 1H NMR (CDCl3): δ 1.43 (t, J=7.1 Hz, 3H), 4.43
(q, J=7.1 Hz, 2H), 7.15-7.16 (m, 1H), 7.31 (d, J = 8.1 Hz, 1H),
7.41 (dd, J = 7.0 Hz, 1H), 7.84 (m, 1H), 9.98 ppm (broad s,
[3H]Colchicine Binding Assay. The reaction mixtures con-
tained 1.0 μM tubulin, 5.0 μM [3H]colchicine, and 5.0 μM
inhibitor and were incubated 10 min at 37 ꢀC. Complete details
were described previously.32
Cell Lines. Methodology for the evaluation of the growth of
human MCF-7 breast carcinoma cells was previously de-
scribed.32 Human HeLa cells (from carcinoma of the uterine
cervix) and HCT116/chr3 cells (from colon carcinoma) were
grown at 37 ꢀC in a humidified atmosphere containing 5%
carbon dioxide in D-MEM (Gibco BRL, UK) supplemented
with 10% fetal calf serum, 4 mM glutamine, 2 mM sodium
pyruvate, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (all
reagents were from Celbio, Italy). For the HCT116/chr3 cells,
the medium also included 500 μg/mL of the selection compound
G418 (Gibco). Cells were trypsinized when subconfluent and
seeded in T75 flasks at a concentration of 105/mL. U937 (human
monocytic leukemia cell line) and J774.1 cells (murine macro-
phage cell line) were cultured in RPMI 1640 medium containing
10% fetal calf serum, 50 μg/mL each of penicillin and strepto-
mycin, and 2 mM glutamine. Human melanoma M14 cells and
human embryonic kidney (HEK) 293 cells were grow at 37 ꢀC in
Dulbecco’s modified Eagle’s medium containing 10 mM glucose
(DMEM-HG) supplemented with 10% fetal calf serum and
50 μg/mL each of penicillin and streptomycin.
disappeared on treatment with D2O, 1H). IR: ν 1690, 3311 cm-1
.
Anal. (C11H10BrNO2 (268.11)).
5-Bromo-1H-indole-2-carboxylic Acid (59). A mixture of 58
(0.25 g, 0.001 mol) in 3 N sodium hydroxide (2 mL) was placed
into the MW cavity (closed vessel mode, Pmax=250 PSI). MW
irradiation at 150 W was used, the temperature being ramped
from 25 to 110 ꢀC. Once 110 ꢀC was reached, taking about 1 min,
the reaction mixture was held at this temperature for 2 min while
stirring. After cooling, the reaction mixture was made acidic
with 3 N hydrochloric acid (pH = 2) and extracted with ethyl
acetate; the organic layer was washed with brine, dried, filtered,
and evaporated to give 59 as a brown solid (0.23 g, 95%), mp
1
188 ꢀC (from ethanol). H NMR (DMSO-d6): δ 7.05 (s, 1H),
7.32 (dd, J = 8.9 Hz, 1H), 7.39 (d, J = 8.8 Hz, 1H), 7.85 (d,
J=2.1 Hz, 1H), 11.94 (broad s, disappeared on treatment with
D2O, 1H), 12.61 ppm (broad s, disappeared on treatment with
D2O, 1H). IR: ν 1652, 2850, 3422 cm-1. Anal. (C9H6BrNO2
(240.05)).
Methyl 5-Bromo-1H-indole-2-carboxylate (60). To a cold
suspension of 59 (3.4 g, 0.014 mol) in anhydrous methanol
(13.4 mL) was added thionyl chloride (1.6 mL) dropwise. The
reaction mixture was stirred at the same temperature for 20 min
and then heated at 65 ꢀC for 2 h under an Ar stream, then
cooled and diluted with water and ethyl acetate. The layers
The P. tridactylis PtK2 cells, the A10 rat embryonic aortic
smooth muscle cells, the human umbilical vein endothelial cells,
and the human aortic smaooth muscle cells were obtained from
the AmericanTypeTissue Collection andgrownas recommended
by the supplier, except that a 5% CO2 atmosphere was used with
all cells.

7526 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
La Regina et al.
Morphological Analysis. Cells grown in T75 flasks and treated
with 100 μM 24 for 24 h were observed with an Olympus IX71
microscope and photographed with a Cool SNAPESES Photo-
metric digital camera.
to A.I.S. V.G. is a Ph.D. student from the University of Pavia
(Dottorato in Scienze biomolecolari e biotecnologie).
Supporting Information Available: Effects of compounds 24,
27-29, 36, 39, and 41 on murine macrophage J744.1 cell
morphology. Elemental analyses of new derivatives 7, 8, 10,
11, 13, 14, 16, 17, 19-23, 25-32, 35, 37-41, and 43-56. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Cell Viability Assay. HeLa and HCT116/chr3 cells. Cells
were seeded in 96-well plates at a density of 1ꢀ103 cells/100 μL
well. Twenty-four hours later, cells were treated with the test
compound (stock solution: 10 mM in DMSO, kept at room
temperature in the dark) used at increasing concentrations
(0.1-100 μM) for 24 h. Cells were harvested at the end of the
treatment or washed with phosphate-buffered saline and in-
cubated in drug-free medium for an additional 24 h. To rule
out a possible effect of the solvent, parallel samples were
incubated in complete medium containing 1% DMSO. At
the end of the treatments, 20 μL of CellTiter96 Aqueous One
Solution Reagent (Promega) was added to each well. The
plates were then incubated for 4 h at 37 ꢀC and analyzed with
a microplate reader (Gio. De Vita, Roma, Italy) at 492 nm.
Experiments were performed in quadruplicate and repeated
three times.
References
(1) Teicher, B. A. Newer cytotoxic agents: attacking cancer broadly.
Clin. Cancer Res. 2008, 14, 1610–1617.
(2) Bhattacharyya, B.; Panda, D.; Gupta, S.; Banerjee, M. Antimitotic
acticity of colchicine and the structural basis for its interaction with
tubulin. Med. Res. Rev. 2008, 28, 155–183.
(3) 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.
(4) Nogales, E.; Whittaker, M.; Milligan, R. A.; Downing, K. H. High-
resolution model of the microtubule. Cell 1999, 96, 79–88.
(5) 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.
(6) 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. Cyclos-
treptin binds covalently to microtubule pores and lumenal taxoid
binding sites. Nat. Chem. Biol. 2007, 3, 117–125.
(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) Beckers, T.; Mahboobi, S. Natural, semisynthetic and synthetic
microtubule inhibitors for cancer therapy. Drugs Future 2003, 28,
767–785.
(9) 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.
(10) 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 polymer-
ization. J. Med. Chem. 2004, 47, 6120–6123.
M14, HEK, U937, and J774.1 cells. Cell viability was deter-
mined using the 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetra-
zolium bromide (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 105 cells/
100 μL well. After 24 h of growth to allow attachment to the
wells, compounds were added at various concentrations (10, 30,
50, 100, 150, and 200 nM). After 24 or 48 h of growth and after
removal of the culture medium, 100 μL/well of medium contain-
ing 1 mg/mL of MTT was added. Cell cultures were further
incubated at 37 ꢀC for 2 h in the dark. The MTT solution was
then removed, and 100 μL DMSO was added to dissolve
formazan crystals. Absorbance at 550 nm was measured using
a plate reader. Experiments were performed in triplicate. As a
control, 0.5% DMSO was added to untreated cells.
Time-Lapse Microscopy. Effect of drug treatment on HEK
and M14 cell morphology was determined by TLM using a
Leica CTR6500 microscope. Images were captured every hour
for a 48 h period. Plates (96-well) were incubated under standard
culture conditions and kept at 37 ꢀC in a 5% carbon dioxide
atmosphere for the observation period (up to 48 h).
(11) De Martino, G.; Edler, M. C.; La Regina, G.; Coluccia, A.;
Barbera, M. C.; Barrow, D.; Nicholson, R. I.; Chiosis, G.; Bran-
cale, 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.
Yeast. S. cerevisiae yeast wild type strain BY4741 (Mat a
his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used. Cells were grown at
28 ꢀC in minimal medium (0.67% yeast nitrogen base without
amino acids) containing 2% glucose (synthetic dextrose, SD)
supplemented with 20 μg/μL of the appropriate nutritional
supplements. A fresh exponential culture (5ꢀ106 cells/mL) of
the yeast were incubated for 18 h at 28 ꢀC with the compound of
interest at 20 μM. The cells were then centrifuged for 5 min at
13000g at 4 ꢀC, washed twice with distilled water, and resus-
pended in SD (1 mL). Cell suspensions (15 μL) were transferred
in 60 μL of fresh SD medium, serially diluted 10-fold, incubated
overnight at 28 ꢀC, and spotted onto YP plates (1% yeast
extract, 2% peptone) supplemented with 2% glucose (YPD).
The plates were incubated at 28 ꢀC for three days before
recording cell growth.
(12) La Regina, G.; Edler, M. C.; Brancale, A.; Kandil, S.; Coluccia, A.;
Piscitelli, F.; Hamel, E.; De Martino, G.; Matesanz, R.; Dıaz, J. F.;
´
Scovassi, A. I.; Prosperi, E.; Lavecchia, A.; Novellino, E.; Artico,
M.; Silvestri, R. New arythioindoles inhibitors of tubulin polymer-
ization. 3. Biological evaluation, SAR and molecular modeling
studies. J. Med. Chem. 2007, 50, 2865–2874.
(13) Tron, G. C.;Pirali, T.;Sorba, G.;Pagliai, F.;Busacca, S.;Genazzani,
A. A. Medicinal chemistry of combretastatin A4: present and future
directions. J. Med. Chem. 2006, 49, 1–12.
(14) Liou, J.-P.; Chang, Y.-L.; Kuo, F.-M.; Chang, C.-W.; Tseng,
H.-Y.; Wang, C.-C.; Yang, Y.-N.; Chang, J.-Y.; Lee, S.-J.; Hsieh,
H.-P. Concise synthesis and structure-activity relationships of
combretastatin A-4 analogues, 1-aroylindoles, and 3-aroylindoles
as novel classes of potent antitubulin agents. J. Med. Chem. 2004,
47, 4247–4257.
(15) Mori, K.; Takaishi, H. Synthetic microbial chemistry. XXII.
Synthesis of monocerin, an antifungal, insecticidal and phytotoxic
heptaketide metabolite of Exserohilum monoceras. Tetrahedron
1989, 45, 1639–1646.
(16) Mitsunobu, O.; Yamada, M. Preparation of esters of carboxylic
and phosphoric acid via quaternary phosphonium salts. Bull.
Chem. Soc. Jpn. 1967, 40, 2380–2382.
Acknowledgment. This research was funded in part by
Istituto Pasteur;Fondazione Cenci Bolognetti, 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 all’Estero. G.L.R. was sup-
ported by a fellowship from FIRC. L.M. thanks Progetti di
ꢀ
ꢀ
Ricerca di Universita, Sapienza Universita di Roma, for her
Contratto di Collaborazione Esterna per Progetti di Ricerca
(Bando 2/2007, Dipartimento di Studi Farmaceutici). This
research was also partially supported by a grant of the
Fondazione Banca del Monte di Lombardia (Pavia, Italy)
(17) Botstein, D.; Chervitz, S. A.; Cherry, J. M. Yeast as a model
organism. Science 1997, 277, 1259–1260.
(18) Almeida, B.; Silva, A.; Mesquita, A.; Sampaio-Marques, B.;
Rodrigues, F.; Ludovico, P. Drug-induced apoptosis in yeast.
Biochim. Biophys. Acta 2008, 1783, 1436–1448.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7527
(19) Zabrocki, P.; Bastiaens, I.; Delay, C.; Bammens, T.; Ghillebert, R.;
Pellens, K.; De Virgilio, C.; Van Leuven, F.; Winderick, J. Phos-
phorylation, lipid raft interaction and traffic of alpha-synuclein in a
yeast model for Parkinson’s disease. Biochim. Biophys. Acta 2008,
1783, 1767–1780.
(25) Tullberg, E.; Schacher, F.; Peters, D.; Frejd, T. Solvent-free Heck-
Jeffery reactions under ball-milling conditions applied to the
synthesis of unnatural amino acids precursors and indoles. Synth-
esis 2006, 7, 1183–1189.
(26) Davis, D. P.; Kirk, K. L.; Cohen, L. A. New synthesis of 2-
nitroimidazoles. J. Heterocycl. Chem. 1982, 19, 253–256.
(27) Molecular Operating Environment (MOE 2007.09). Chemical
Computing Group, Inc. Montreal, Quebec, Canada, http://www.
chemcomp.com.
(20) Cassidy-Stone, A.; Chipuk, J. E.; Ingerman, E.; Song, C.; Yoo,
C.; Kuwana, T.; Kurth, M. J.; Shaw, J. T.; Hinshaw, J. E.; Green,
D. R.; Nunnari, J. Chemical inhibition of the mitochondrial divi-
sion dynamin reveals its role in Bax/Bak-dependent mitochondrial
outer membrane permeabilization. Dev. Cell 2008, 14, 193–204.
(21) Nguyen, T. L.; McGrath, C.; Hermone, A. R.; Burnett, J. C.;
Zaharevitz, D. W.; Day, B. W.; Wipf, P.; Hamel, E.; Gussio, R. A
common pharmacophore for a diverse set of colchicine site inhibitors
using a structure-based approach. J. Med. Chem. 2005, 48, 6107–6116.
(22) Dupeyre, G.; Chabot, G. G.; Thoret, S.; Cachet, X.; Seguin, J.;
Guenard, D.; Tillequin, F.; Scherman, D.; Koch, M.; Michel, S.
Synthesis and biological evaluation of (3,4,5-trimethoxyphe-
nyl)indol-3-ylmethane derivatives as potential antivascular agents.
Bioorg. Med. Chem. 2006, 14, 4410–4426.
(28) FlexX 3.0; BioSolveIT GmbH: Sankt Augustin, Germany; http://www.
biosolveit.de. Accessed on 11/12/2008.
(29) Code “fragment_rmsd.svl” obtained from SVL exchange website:
SVL Exchange: An SVL Code Exchange Site for the MOE User
Community; Chemical Computing Group, Inc.: Montreal, Canada,
2004; http://svl.chemcomp.com. Accessed on 11/12/2008.
(30) Zodiac 0.5b; http://www.zeden.org. Accessed on 11/12/2008.
(31) Hamel, E. Evaluation of antimitotic agents by quantitative com-
parisons of their effects on the polymerization of purified tubulin.
Cell Biochem. Biophys. 2003, 38, 1–21.
(23) Liou, J.-P.; Wu, C.-Y.; Hsieh, H.-P.; Chang, C.-Y.; Chen, C.-M.;
Kuo, C.-C.; Chang, J.-Y. 4- and 5-Aroylindoles as novel classes of
potent antitubulin agents. J. Med. Chem. 2007, 50, 4548–4552.
(24) Jia, Y.; Zhu, J. Palladium-catalyzed, modular synthesis of highly
functionalized indoles and tryptophans by direct annulation of
substituted o-haloanilines and aldehydes. J. Org. Chem. 2006, 71,
7826–7834.
(32) 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 anti-
mitotic agent, with tubulin and effects of analogs on the
growth of MCF-7 breast cancer cells. Mol. Pharmacol. 1998, 53,
62–76.