New arylthioindoles: Potent inhibitors of tubulin polymerization. 2. Structure-activity relationships and molecular modeling studies

Article abstract of DOI:10.1021/jm050809s

Arylthioindoles (ATIs) that possess a 3-methoxyphenylthio or a 3,5-dimethoxyphenylthio moiety at position 2 of the indole ring were effective tubulin assembly inhibitors, but weak inhibitors of MCF-7 cell growth. ATIs bearing a 3-(3,4,5-trimethoxyphenyl)thio moiety were potent tubulin polymerization inhibitors, with IC₅₀s in the 2.0 (35) to 4.5 (37) μM range. They also inhibited MCF-7 cell growth at nanomolar concentrations. The 3,4,5-trimethoxy substituted ATIs showed potencies comparable to those of the reference compounds colchicine and combretastatin A-4 in both tubulin assembly and cell growth inhibition assays. Dynamics simulation studies correlate well with the observed experimental data. Furthermore, from careful analysis of the biological and in silico data, we can now hypothesize a basic pharmacophore for this class of compounds.

Full text of DOI:10.1021/jm050809s

J. Med. Chem. 2006, 49, 947-954
947
New Arylthioindoles: Potent Inhibitors of Tubulin Polymerization. 2. Structure-Activity
Relationships and Molecular Modeling Studies
Gabriella De Martino,† Michael C. Edler,§ Giuseppe La Regina,† Antonio Coluccia,† Maria Chiara Barbera,‡ Denise Barrow,|
Robert I. Nicholson,| Gabriela Chiosis, Andrea Brancale,‡ Ernest Hamel,§ Marino Artico,† and Romano Silvestri*,†
Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, UniVersita` di Roma “La Sapienza”, Piazzale Aldo Moro 5,
I-00185 Roma, Italy, Welsh School of Pharmacy, Cardiff UniVersity, King Edward VII AVenue, Cardiff, CF10 3XF, UK, Program in Cell
Biology, Memorial Sloan-Kettering Cancer Center, 1275 York AVenue, New York, New York 10021, 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, and TenoVus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff UniVersity, King Edward
VII AVenue, Cardiff, CF10 3XF, UK
ReceiVed August 15, 2005
Arylthioindoles (ATIs) that possess a 3-methoxyphenylthio or a 3,5-dimethoxyphenylthio moiety at position
2 of the indole ring were effective tubulin assembly inhibitors, but weak inhibitors of MCF-7 cell growth.
ATIs bearing a 3-(3,4,5-trimethoxyphenyl)thio moiety were potent tubulin polymerization inhibitors, with
IC50s in the 2.0 (35) to 4.5 (37) µM range. They also inhibited MCF-7 cell growth at nanomolar concentrations.
The 3,4,5-trimethoxy substituted ATIs showed potencies comparable to those of the reference compounds
colchicine and combretastatin A-4 in both tubulin assembly and cell growth inhibition assays. Dynamics
simulation studies correlate well with the observed experimental data. Furthermore, from careful analysis
of the biological and in silico data, we can now hypothesize a basic pharmacophore for this class of
compounds.
Chart 1
Introduction
Anticancer therapy based on microtubule-targeting agents is
receiving growing attention. Tubulin heterodimers, the major
component of microtubules, are the molecular target of these
agents. Inhibition of tubulin polymerization or interfering with
microtubule disassembly disrupts several cellular functions, in-
cluding cell motility and mitosis.1-3 The former group of agents
includes such compounds as colchicine (1), combretastatin A-4
(2a; CSA4), and Catharanthus alkaloids, such as vinblastine,
vincristine, and vinorelbine. The microtubule stabilizing drugs
include the clinically important taxoids paclitaxel and docetaxel,
as well as more recently discovered natural products, such as
the epothilones, discodermolide, and eleutherobin. As a group,
drugs that bind to either unpolymerized tubulin or tubulin
polymer interfere both with cell division and interphase functions
that require a normal microtubule cytoskeleton. Cells treated
with these agents invariably undergo apoptosis.4
Since clinically useful antitubulin drugs all have problems
with toxicity, drug resistance, and bioavailability, there is a
continuing effort to find new compounds that might be safer or
more effective.2,5 Several compounds are currently in ongoing
clinical trials, for example various epothilones and taxoids, re-
presenting the stabilizer class, and the destabilizer combretastatin
A-4 phosphate (2b), ZD 61266 (3), the semisynthetic vinca
alkaloid vinflunine,7 the naturally occurring peptide dolastatin
10,8 the sulfonamide E7010,9 and T138067/T900607.3
and the growth of MCF-7 human breast carcinoma cells14 (for
general formula of ATIs, see structure 4). The mechanism of
action was through binding at the colchicine site on â-tubulin.
In our preliminary work, structure-activity relationship (SAR)
analysis highlighted four determinant structural requirements:
(A) the ester function at position 2 of the indole; (B) the
3-arylthio group; (C) the sulfur atom bridge; (D) the substituent
at position 5 of the indole, especially for the inhibition of MCF-7
cell growth.14 Pursuing our research project, here we describe
the synthesis, biological evaluation, and molecular modeling
studies of new ATI derivatives (Chart 2).
Several indoles were found to be effective as inhibitors of
tubulin assembly.10-13 We recently discovered arylthioindoles
(ATIs) as a new class of potent inhibitors of tubulin assembly
Chemistry
* Corresponding author. Phone +39 06 4991 3800, Fax +39 06 491
491, E-mail: romano.silvestri@uniroma1.it.
† Universita` di Roma.
Synthesis and structures of new ATI derivatives are depicted
in Scheme 1 and Table 1, respectively. Compounds 12, 13, 14,
17, 19, 28, 31-34, and 38 were synthesized by treating
appropriate indole-2-carboxylates with N-arylthiosuccinimides
in the presence of boron trifluoride diethyl etherate in anhydrous
‡ Cardiff University.
§ National Cancer Institute.
Memorial Sloan-Kettering Cancer Center.
| Tenovus Centre for Cancer Research.
10.1021/jm050809s CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/19/2006

948 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Chart 2. SAR Study of ATIs
De Martino et al.
cell growth (IC50’s in 0.15 to 0.33 µM range). Thus, for example,
methyl 3-[(3-methoxyphenyl)thio]-5-methoxy-1H-indole-2-car-
boxylate (33, IC50 ) 3.1 µM) was as active as colchicine as an
inhibitor of tubulin assembly, and methyl 3-[(3-methoxyphenyl)-
thio]-5-chloro-1H-indole-2-carboxylate (19, IC50 ) 1.8 µM) was
1.8 times more potent than colchicine and slightly superior to
CSA4. These potent tubulin assembly inhibitors, however, were
more than 10-fold less inhibitory than the reference compounds
1 (colchicine) and 2a (CSA4) as inhibitors of MCF-7 cell
growth.
CH2Cl2, as we previously reported.15 Compounds 16, 27, 36,
and 41 were obtained following a two-step procedure involving
addition of 3,4,5-trimethoxylthiophenol to a solution of N-
chlorosuccinimide (NCS) at -78 °C, and this mixture was
treated with the indole-2-carboxylate while the reaction tem-
perature was warmed to 0 °C over 1 h. Compound 25 was
prepared by treatment of indole-2-carboxylic acid with 1,1′-
(3,5-dimethoxyphenyl)disulfide in the presence of NaH. The
crude acid was transformed into the corresponding methyl ester
by reaction with trimethylsilyldiazomethane (TMSDM) at room
temperature for 30 min. In the case of compounds 37 and 40,
the crude acids were transformed into the corresponding methyl
esters by refluxing in methanol in the presence of thionyl
chloride for 48 h under a stream of anhydrous argon. This
procedure was also used for the preparation of esters 5-11
starting from 3-phenylthio-1H-indole-2-carboxylic acid15 and
appropriate alcohols. Oxidation of sulfur derivatives 17, 19, 21,
29, and 38 with 3-chloroperoxybenzoic acid (MCPBA) furnished
the corresponding sulfones 18, 20, 22, 30, and 39, respectively.
Introduction of a second methoxy group at position 5 of the
arylthio moiety of 19 produced methyl 3-[(3,5-dimethoxyphe-
nyl)thio]-5-chloro-1H-indole-2-carboxylate (25). Compound 25
differed little from 19 in its inhibitory effect on tubulin assembly,
but 25 was almost 10-fold more cytotoxic. Worthy of note,
replacement of the 3-(3,5-dimethoxyphenyl)thio group of 25
with a 3-(3,5-dimethylphenyl)thio moiety (23) produced an
inactive compound. The ethyl ester analogue of 23, compound
24, was also essentially inactive.
ATI derivatives 15, 16, 26, 27, 35, 36, 37, 40, and 41 bear a
3-(3,4,5-trimethoxyphenyl)thio moiety. The first seven were all
highly active as inhibitors of tubulin polymerization, with IC50
values in the 2.0 (35) to 4.5 (37) µM range. These values were
comparable to those of the reference compounds colchicine (IC50
) 3.2 µM) and CSA4 (IC50 ) 2.2 µM). The 3,4,5-trimethoxy
substitution of the phenylthio group was found to be of crucial
importance for potent MCF-7 cell growth inhibition. In fact,
with the sole exception of the 3,5-dimethoxy derivative 25, only
compounds bearing the 3-(3,4,5-trimethoxyphenyl)thio moiety
had IC50 values of less than 50 nM against the MCF-7 cells,
with the most potent ATI being compound 35 (compare 15 (IC50
) 25 nM), 16 (IC50 ) 40 nM), 26 (IC50 ) 42 nM), and 35
(IC50 ) 13 nM)). With the MCF-7 cells, methyl esters were
about 2 to 3 times more active than the corresponding ethyl
esters (compare 15 with 16, 26 with 27, and 35 with 36). A
similar difference was not observed in effects on tubulin
assembly. We also obtained some data regarding effects of
susbtituents at position 5 of the indole ring. Order of activity,
at least for inhibition of cell growth, was H3CO (35) > H (15)
> Cl (26) > NO2 (37), thus suggesting a correlation between
electron-donating effect and cytotoxicity.
Results and Discussion
Table 2 summarizes the biological data for inhibition of
tubulin polymerization, colchicine binding to tubulin (more
active compounds only), and growth of MCF-7 human breast
carcinoma cells by the indoles 5-41 in comparison with data
obtained with the reference compounds colchicine (1) and CSA4
(2a). Biological evaluation of selected ATIs against small cell
lung cancer (SCLC) carcinoma cells is also reported.
Previously, we found a 2- to 4-fold increase in the inhibitory
effects of ATI derivatives on tubulin polymerization by intro-
ducing either a methoxycarbonyl (IC50 ) 8.2 µM) or an
ethoxycarbonyl (IC50 ) 4.4 µM) function at position 2 of the
indole ring of 3-phenylthio-1H-indole.14 We therefore evaluated
the effect of additional esters with either a linear or a branched
alkoxy chain (compounds 5-11) at this position. Only the
isopropyl ester of 3-(phenylthio)-1H-indole-2-carboxylic acid
(6) had any activity (IC50 ) 18 µM). All other tested compounds
were inactive in both the tubulin polymerization and MCF-7
cell assays.
Changing the position of the methoxy group on the indole
from position 5 to 4 or 6 led to loss of activity in inhibiting
tubulin assembly (compare 29 with 31 and 28; all three
compounds were inactive against the MCF-7 cells). Similarly,
introduction of an additional methoxy group at position 6 of
35 or 36 to give compounds 40 and 41 resulted in a dramatic
loss of activity. Finally, as observed previously,14 oxidation of
the sulfur atom to the sulfone always resulted in a large loss of
activity (compare 17 with 18, 19 with 20, 29 with 30, and 38
with 39).
The tubulin assembly inhibitory activity of ATIs bearing a
single methoxy group on the 3-arylthio moiety was strongly
dependent on the position of the methoxy on the phenyl ring.
Invariably, within the cohort of compounds synthesized, the
greatest activity occurred when the methoxy group was located
meta, the least when located para, to the bridging atom (compare
13 with 12 and 14, 19 with 17 and 21, 33 with 32 and 34).
Biological data against the SCLC cells showed good agree-
ment with the data for the MCF-7 cells, although, in this case,
compound 25 appeared to be the most active agent in the series.
Due to structural similarly of the ATIs with the Hsp90
inhibitor PU3,16 we evaluated compound 26 for binding to
Hsp90. We found that 26 did not compete with geldanamycin
for binding to Hsp90R, and consequently it did not induce the
degradation of Her2. As would be expected for an agent with
antitubulin activity, however, compound 26 caused MDA-MB-
468 breast cancer cells to accumulate in mitotic arrest, as
measured with the Tg3 antibody (Tg3 recognizes p-nucleolin,
which is highly expressed during mitosis).
This inhibitory effect was unpredictably affected by substit-
uents at position 5 of the indole ring (compare the ethyl esters
12 and 32 and also the methyl ester 17, the ethyl esters 13 and
33 and also the methyl ester 19).
Notably, of the compounds with a single methoxy group on
the phenyl ring, potent inhibitory activity against tubulin
assembly was observed with compounds 13, 17, 19, and 33,
and all except 17 were reasonably effective inhibitors of MCF-7

Arylthioindoles Inhibitors of Tubulin Polymerization
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 949
Scheme 1a
a Reagents and reaction conditions: (a) (12, 13, 14, 17, 19, 28, 31-34 and 38) N-[(R2-R5)PhS]succinimide, BF3.Et2O, anhydrous CH2Cl2, r.t., 1.5 h, then
45 °C, 2 h; (b) (16, 27, 36, and 41) (i) 3,4,5-(MeO)3PhSH, NCS, CH2Cl2, -78 °C to 0 °C, 1 h; (c) (25) (i) [3,5-(MeO)2PhS]2, NaH, anhydrous DMF, 50 °C,
overnight, anhydrous argon stream; (ii) TMSDM, CH3OH/CH2Cl2, r.t., 30 min; (d) (5-11, 37, and 40) (i) [(R2-R5)PhS]2, NaH, anhydrous DMF, 50 °C,
overnight, anhydrous argon stream; (ii), SOCl2, ROH, reflux, 48 h, anhydrous argon stream; (e) (18, 20, 22, 30, and 39) MCPBA (2.5 eq), CHCl3, r.t., 1 h.
Table 1. Structure of Arythioindoles 5-41
Table 2. Inhibition of Tubulin Polymerization, Growth of MCF-7
Human Breast Carcinoma Cells, Colchicine Binding, and Growth of
SCLC Cells by Compounds 5-41
tubulina
MCF-7b
SCLCd
IC50 ( SD IC50 ( SD inhibition colchicine IC50 ( SD
compd
(µM)
(nM)
bindingc (% ( SD)
(nM)
5
6
7
8
>40
>2.5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
18 ( 2
>40
>2.5
>2.5
compd
R1
R2
R3
R4
R5
R6
R7
R8
X
>40
>2.5
9
>40
>2.5
5
6
7
8
COO-n-Pr
COO-i-Pr
COO-n-Bu
COO-s-Bu
COO-t-Bu
COO-i-Pe
COOCH2Ph
COOEt
COOEt
COOEt
COOMe
COOEt
COOMe
COOMe
COOMe
COOMe
COOMe
COOMe
COOMe
COOEt
COOMe
COOMe
COOEt
COOMe
COOMe
COOMe
COOMe
COOEt
H
H
H
H
H
H
H
OMe
H
H
H
H
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
S
S
S
S
S
S
S
S
S
S
S
S
S
SO2
S
SO2
S
SO2
S
S
S
S
S
S
S
SO2
S
S
S
S
S
S
S
S
10
11
12
13
14
15e
16
17
18
19
20
21e
22
23e
24e
25
26e
27
28
29e
30
31
32
33
34
35e
36
37
38
39
40
41
Col.e
>40
>2.5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
>40
>2.5
12 ( 2
2.9 ( 0.3
>40
>2.5
150 ( 90
>2.5
48 ( 7
-
585 ( 50
9
>10000
10
11
12
13
14
15a
16
17
18
19
20
21a
22
23b
24b
25
26a
27
28
29a
30
31
32
33
34
35a
36
37
38
39
40
41
2.9 ( 0.1
2.9 ( 0.2
4.2 ( 0.6
>40
25 ( 1
40 ( 2
1300 ( 400
>2.5
74 ( 2
52 ( 3
51 ( 3
84 ( 5
34 ( 6
7000 ( 300
OMe
H
OMe OMe OMe
OMe OMe OMe
H
H
OMe
OMe
H
H
OMe
-
-
1.8 ( 0.2
31 ( 4
>40
330 ( 40
>2.5
53 ( 4
-
-
-
-
-
3200 ( 800
>10000
>2.5
>10000
H
H
H
H
OMe
OMe
H
H
H
H
H
H
H
H
H
Me
Me
OMe
>40
>2.5
-
-
-
>40
>2.5
>40
1200 ( 200
34 ( 10
42 ( 10
110 ( 20
>2.5
2.2 ( 0.2
2.5 ( 0.3
2.2 ( 0.2
>40
82 ( 2
18 ( 1
H
57 ( 2
216 ( 17
Me
Me
OMe
OMe OMe OMe
OMe OMe OMe
H
H
H
H
H
OMe
H
OMe OMe OMe
OMe OMe OMe
OMe OMe OMe
H
H
53 ( 6
93 ( 10
-
-
-
-
-
6.2 ( 0.3
>40
>40
16 ( 0.5
3.1 ( 0.2
>40
2.0 ( 0.2
2.4 ( 0.2
4.5 ( 0.1
14 ( 1
>40
>40
22 ( 0.7
3.2 ( 0.4
>2.5
30 ( 6
-
-
-
39 ( 3
-
>2.5
>2.5
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
350 ( 60
280 ( 100
>2.5
2200 ( 200
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
NO2
OMe OMe
OMe OMe SO2
OMe OMe
OMe OMe
584 ( 40
>10000
13 ( 3
46 ( 3
120 ( 40
>2.5
90 ( 1
47 ( 2
71 ( 2
-
-
-
-
-
-
-
-
COOEt
COOEt
COOMe
COOEt
COOMe
COOEt
COOEt
COOMe
COOEt
32 ( 2
-
-
-
-
>2.5
1600 ( 400
1000 ( 200
13 ( 3
17 ( 10
H
H
H
H
-
H
H
H
CSA4e 2.2 ( 0.2
97 ( 0.5
OMe OMe OMe
OMe OMe OMe
S
S
a Inhibition of tubulin polymerization. b Inhibition of growth of MCF-7
human breast carcinoma cells. c Inhibition of [3H]colchicine binding.
d Inhibition of growth of SCLC cells. e Data from ref 14.
a Ref 14. b Ref 15.
Molecular Modeling
predictions. Some docking results were very similar to the
proposed binding mode reported recently,14 but, unfortunately,
none of the scoring results correlated with the biological data
(data not shown). In particular, the scoring results for the
p-methoxyphenyl analogues were often similar to or better than
the corresponding results for the trimethoxyphenyl compounds.
However, FlexX was able to identify an alternate possible
binding conformation for this class of compounds compared
To investigate the structural basis of the SAR that emerged
from the biological results, we carried out docking studies on
the entire series of compounds reported in this paper, using the
FlexX module included in SYBYL.17 The results were evaluated
using the built in scoring function as well as the functions
included in the CScore module, with the aim of finding a good
correlation between the experimental results and the in silico

950 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
De Martino et al.
Figure 1. Two possible binding modes of 36. In green the previously
reported conformation (old pose); in red the current proposed confor-
mation (new pose).
Figure 3. Proposed binding mode for colchicine. Leu248 and Leu255
in gray, Cys241 in yellow, Thr179 and Lys352 in stick representation.
These results encouraged us to extend our molecular dynamics
studies to other compounds and, in particular, to the p-, m-,
and o-methoxyphenyl analogues (32, 33, 34). We also performed
the simulation under the same conditions on colchicine to have
a better understanding of its binding mode with tubulin. For
the latter study, we modified the structure of the N-deacetyl-
N-(2-mercaptoacetyl)colchicine cocrystallized with tubulin, and
the resulting complex was energy minimized before the mo-
lecular dynamics experiment was performed.
Colchicine established one hydrogen bond between the
carbonyl group of ring C and Lys352 and another between the
NH group and Thr179 (Figure 3), the same residues involved
in the binding of 36 (Figure 2). During the simulation, the amide
moiety changed its orientation, losing the hydrogen bond with
Thr179 and forming another one between the amide carbonyl
group and the hydroxyl group of Ser178. The trimethoxy ring
A remains placed in a hydrophobic pocket where, in particular,
two leucine residues (Leu248 and Leu255) interact strongly with
the aromatic ring. Cys241, a key residue for the binding and
biological activity of many colchicine analogues,19 is in close
proximity to the C-3 methoxy group of ring A. The average
value of the interaction potential energy for colchicine was
-71.6 kcal/mol.
The para-substituted arylthioindole 34, on the other hand, does
not establish a hydrogen bond with Thr179 (Figure 4). The
interaction with Lys352 remains stable during the simulation
time, while the aromatic ring does not establish any contact
with Cys241. The energy value obtained for binding, -49.8
kcal/mol, is in agreement with the poor activity observed in
the biological experiments. This is ∼15 kcal/mol higher than
the value obtained for 36, indicating a reduced binding affinity
for 34 relative to 36.
Figure 2. Proposed binding mode for 36. Lys352 in blue, Thr179 in
red, Cys241 in yellow.
with the one reported previously (Figure 1). In this new pose,
the indole moiety still forms a hydrogen bond with Thr179
(residue number based on the crystal structure used), but now
the ester group is positioned deep in the binding pocket. In
addition, the carbonyl of the ATI ester group forms another
hydrogen bond with Lys352 (Figure 2).
In comparing these two possible binding modes, the scoring
functions employed were not able to clearly indicate which
would be the preferred conformation. To solve this problem,
we carried out molecular dynamics simulations (MD) on the
tubulin-ligand complexes to evaluate the potential energy of
the binding of the two different poses of 36 in the colchicine
binding site. The calculations were performed using the MOE
(Molecular Operating Environment)18 software package, allow-
ing free movement of the residues within a 15 Å distance of
the ligand, keeping the rest of the protein fixed. This site was
soaked in water, and the simulation was run in the NVT (number
of particles, volume, and temperature of the system are kept
constant during the simulation) environment, for a total of 600
ps at 300 K. The states generated during the first 100 ps were
not considered in evaluating the results.
The results obtained for the ortho- and meta-substituted
arylthioindoles (32 and 33) also correlated well with the
experimental data. Both compounds established hydrogen bonds
with Thr179 and Lys352 through the indole ring. The phenyl
ring of compound 33 assumes a similar orientation as the
trimethoxy ring of 36, but, in the case of compound 32, the
o-methoxy aryl moiety is almost orthogonal to the corresponding
aromatic ring of 36 (Figure not shown). The energy values
obtained for 32 and 33 are in agreement with these observations
and with the experimental results (Table 3).
The interaction potential energy values calculated by the
dynamics simulations revealed a difference of almost 20 kcal/
mol between the two conformations (average values: -64.7 kcal/
mol for the new pose, -44.9 kcal/mol for the old pose),
suggesting that 36 would bind with the indole moiety oriented
as in Figure 2.

Arylthioindoles Inhibitors of Tubulin Polymerization
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 951
Method A. General Procedure for the Synthesis of Com-
pounds 12, 13, 14, 17, 19, 28, 31-34, and 38. Example. Ethyl
3-[(2-Methoxyphenyl)thio]-1H-indole-2-carboxylate (12). Boron
trifluoride diethyl etherate (0.067 g, 0.06 mL, 0.00047 mol) was
added to a mixture of ethyl 1H-indole-2-carboxylate (0.53 g, 0.0028
mol), N-(2-methoxyphenylthio)succinimide (0.57 g, 0.0024 mol),
and anhydrous dichloromethane (20 mL) under a dry argon
atmosphere. After stirring at room temperature for 2 h, boron
trifluoride diethyl etherate (0.27 g, 0.24 mL, 0.002 mol) was added,
and the reaction was heated at 45 °C for 2 h. After cooling, the
reaction mixture was diluted with chloroform and brine while being
shaken. The organic layer was separated, washed with a saturated
solution of sodium hydrogen carbonate and with brine, and dried.
The solvent was evaporated to give a residue that was purified by
silica gel column chromatography (ethyl acetate-n-hexane 1:3 as
eluent) to afford 12, yield 26%, mp 164-166 °C (from ethanol).
Anal. Calcd. (C18H17NO3S (327.40)) C, H, N, S.
Ethyl 3-[(3-methoxyphenyl)thio]-1H-indole-2-carboxylate (13)
was prepared as 12 using N-(3-methoxyphenylthio)succinimide.
Yield 23%, mp 101-104 °C (from ethanol).20
Ethyl 3-[(4-methoxyphenyl)thio]-1H-indole-2-carboxylate (14)
was synthesized as 12 using N-(4-methoxyphenylthio)succinimide.
Yield 15%, mp 118-120 °C (from ethanol). Anal. Calcd. (C18H17-
NO3S (327.40)) C, H, N, S.
Figure 4. Proposed binding mode for 34. Lys352 in blue, Thr179 in
red, Cys241 in yellow.
Table 3. Comparison between Average Potential Energy of Interaction
(Uab) and Inhibition of Tubulin Polymerization for Compounds 32, 33,
34, and 36
compd
U
ab (kcal/mol)
tubulin IC50 ( SD (µM)
Methyl 5-chloro-3-[(2-methoxyphenyl)thio]-1H-indole-2-car-
boxylate (17) was synthesized as 12 using methyl 5-chloro-1H-
indole-2-carboxylate and N-(2-methoxyphenylthio)succinimide. Yield
17%, mp 222-226 °C (from ethanol). Anal. Calcd. (C17H14ClNO3S
(347.82)) C, H, Cl, N, S.
32
33
34
36
-57.6
-60.1
-49.8
-64.7
16 ( 0.5
3.1 ( 0.2
>40
2.4 ( 0.2
Methyl 5-chloro-3-[(3-methoxyphenyl)thio]-1H-indole-2-car-
boxylate (19) was synthesized as 12 using methyl 5-chloro-1H-
indole-2-carboxylate and N-(3-methoxyphenylthio)succinimide. Yield
17%, mp 160-163 °C (from ethanol). Anal. Calcd. (C17H14ClNO3S
(347.82)) C, H, Cl, N, S.
Methyl 6-methoxy-3-(phenylthio)-1H-indole-2-carboxylate (28)
was synthesized as 12 using methyl 6-methoxy-1H-indole-2-
carboxylate and N-(phenylthio)succinimide. Yield 5%, mp 120-
124 °C (from ethanol). Anal. Calcd. (C17H15NO3S (313.38)) C, H,
N, S.
Methyl 4-methoxy-3-(phenylthio)-1H-indole-2-carboxylate (31)
was synthesized as 12, using methyl 4-methoxy-1H-indole-2-
carboxylate and N-(phenylthio)succinimide. Yield 40%, mp 115-
118 °C (from ethanol). Anal. Calcd. (C17H15NO3S (313.38)) C, H,
N, S.
Ethyl 5-methoxy-3-[(2-methoxyphenyl)thio]-1H-indole-2-car-
boxylate (32) was synthesized as 12 using ethyl 5-methoxy-1H-
indole-2-carboxylate and N-(2-methoxyphenylthio)succinimide. Yield
35%, mp 158 °C (from ethanol). Anal. Calcd. (C19H19NO4S
(357.43)) C, H, N, S.
Ethyl 5-methoxy-3-[(3-methoxyphenyl)thio]-1H-indole-2-car-
boxylate (33) was synthesized as 12 using ethyl 5-methoxy-1H-
indole-2-carboxylate and N-(3-methoxyphenylthio)succinimide. Yield
31%, mp 104-107 °C (from ethanol). Anal. Calcd. (C19H19NO4S
(357.43)) C, H, N, S.
Ethyl 5-methoxy-3-[(4-methoxyphenyl)thio]-1H-indole-2-car-
boxylate (34) was synthesized as 12 using ethyl 5-methoxy-1H-
indole-2-carboxylate and N-(4-methoxyphenylthio)succinimide. Yield
54%, mp 140-145 °C (from ethanol). Anal. Calcd. (C19H19NO4S
(357.43)) C, H, N, S.
Ethyl 5,6-dimethoxy-3-(phenylthio)-1H-indole-2-carboxylate
(38) was synthesized as 12 using ethyl 5,6-dimethoxy-1H-indole-
2-carboxylate and N-(phenylthio)succinimide. Yield 35%, mp 137-
139 °C (from ethanol). Anal. Calcd. (C19H19NO4S (357.42)) C, H,
N, S.
Method B. General Procedure for the Synthesis of Com-
pounds 16, 27, 36, and 41. Example. Ethyl 3-[(3,4,5-Trimethoxy-
phenyl)thio)]-1H-indole-2-carboxylate (16). 3,4,5-Trimethoxy-
benzenethiol21 (1.87 g, 0.0093 mol) was added to a solution of NCS
(1.26 g, 0.0094 mol) in anhydrous dichloromethane (74 mL) at -78
°C. The reaction was warmed to 0 °C over 15 min, and then a
solution of ethyl 1H-indole-2-carboxylate (1.5 g, 0.008 mol) in the
Conclusions
ATI derivatives that possess a 3-methoxyphenylthio or a 3,5-
dimethoxyphenylthio moiety at position 2 of the indole ring
were potent tubulin inhibitors but weak inhibitors of MCF-7
cell growth. On the other hand, ATIs bearing the 3-(3,4,5-
trimethoxyphenyl)thio moiety were not only potent tubulin
inhibitors, with potencies comparable to those of the reference
compounds colchicine (1) and CSA4 (2a), but also potent
inhibitors of cell growth. Conversely, the 4-methoxyarylthio
substitution was detrimental for inhibition of both tubulin
polymerization and MCF-7 cell growth.
The results obtained from the dynamics simulation correlate
well with the observed experimental data. The agreement
between the SAR findings and the modeling studies increase
our confidence in the binding model we have obtained, and these
results may enable us to design more selective and potent
analogues.
Experimental Section
Chemistry. Melting points (mp) were determined on a Bu¨chi
510 apparatus and are uncorrected. Infrared spectra (IR) were run
on a SpectrumOne FT 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 Merck (70-230
mesh). Aluminum oxide TLC cards from Fluka (aluminum oxide
precoated aluminum cards with fluorescent indicator at 254 nm)
and silica gel TLC cards from Fluka (silica gel precoated aluminum
cards with fluorescent indicator 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. Concentration and evaporation
of the solvent after reaction or extraction was carried out on a Bu¨chi
Rotavapor rotary evaporator operating at reduced pressure. El-
emental analyses were found within (0.4% of the theoretical values.

952 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
De Martino et al.
same solvent (15 mL) was added. The reaction mixture was stirred
at 0 °C for 1 h and concentrated under reduced pressure. The residue
was suspended in water (150 mL) and extracted with ethyl acetate,
and the organic layer was washed with brine and dried. Removal
of the solvent furnished a crude product that was purified by silica
gel column chromatography (ethyl acetate-n-hexane 1:1 as eluent)
to give 16, yield 1%, mp 90-95 °C (from ethanol). Anal. Calcd.
(C20H21NO5S (387.46)) C, H, N, S.
Ethyl 5-chloro-3-[(3,4,5-trimethoxyphenyl)thio)]-1H-indole-
2-carboxylate (27) was synthesized as 16 using ethyl 5-chloro-
1H-indole-2-carboxylate. Yield 6%, mp 110-120 °C (from etha-
nol). Anal. Calcd. (C20H20ClNO5S (421.90)) C, H, Cl, N, S.
Isopropyl 3-(phenylthio)-1H-indole-2-carboxylate (6) was
synthesized as 5 using iso-propyl alcohol. Yield 50%, mp 146 °C
(from ethanol). Anal. Calcd. (C18H17NO2S (311.40)) C, H, N, S.
n-Butyl 3-(phenylthio)-1H-indole-2-carboxylate (7) was syn-
thesized as 5 using n-butyl alcohol. Yield 25%, mp 138 °C (from
ethanol). Anal. Calcd. (C19H19NO2S (325.43)) C, H, N, S.
sec-Butyl 3-(phenylthio)-1H-indole-2-carboxylate (8) was syn-
thesized as 5 using sec-butyl alcohol. Yield 20%, mp 145 °C (from
ethanol). Anal. Calcd. (C19H19NO2S (325.43)) C, H, N, S.
tert-Butyl 3-(phenylthio)-1H-indole-2-carboxylate (9) was
synthesized as 5 using tert-butyl alcohol. Yield 12%, mp 143-
147 °C (from ethanol). Anal. Calcd. (C19H19NO2S (325.43)) C, H,
N, S.
Ethyl 5-methoxy-3-[(3,4,5-trimethoxyphenyl)thio]-1H-indole-
2-carboxylate (36) was synthesized as 16 using ethyl 5-methoxy-
1H-indole-2-carboxylate. Yield 4%, mp 123-128 °C (from etha-
nol). Anal. Calcd. (C21H23NO6S (417.48)) C, H, N, S.
iso-Pentyl 3-(phenylthio)-1H-indole-2-carboxylate (10) was
synthesized as 5 using iso-pentyl alcohol. Yield 21%, mp 124-
128 °C (from ethanol). Anal. Calcd. (C20H21NO2S (339.46)) C, H,
N, S.
Ethyl 5,6-dimethoxy-3-[(3,4,5-trimethoxyphenyl)thio]-1H-in-
dole-2-carboxylate (41) was synthesized as 16 using ethyl 5,6-
dimethoxy-1H-indole-2-carboxylate. Yield 10%, mp 140-146 °C
(from ethanol). Anal. Calcd. (C22H25NO7S (447.51)) C, H, N, S.
Benzyl 3-(phenylthio)-1H-indole-2-carboxylate (11) was syn-
thesized as 5 using benzyl alcohol. Yield 20%, mp 135-138 °C
(from ethanol). Anal. Calcd. (C22H17NO2S (359.45)) C, H, N, S.
Methyl 5-nitro-3-[(3,4,5-trimethoxyphenyl)thio]-1H-indole-2-
carboxylate (37) was synthesized as 5 using 5-nitro-1H-indole-2-
carboxylic acid and 1,1′-(3,4,5-trimethoxyphenyl)disulfide. Trans-
formation of 5-nitro-3-[(3,4,5-trimethoxyphenyl)thio]-1H-indole-
2-carboxylic acid into 37 was achieved by heating at reflux in
methanol in the presence of thionyl chloride. The crude product
was purified by silica gel column chromatography (ethyl acetate-
n-hexane 1:2 as eluent), yield 3%, mp 194-198 °C (from ethanol).
Anal. Calcd. (C19H18N2O7S (418.43)) C, H, N, S.
Methyl 5,6-dimethoxy-3-[(3,4,5-trimethoxyphenyl)thio]-1H-
indole-2-carboxylate (40) was synthesized as 5 using 5,6-dimethoxy-
1H-indole-2-carboxylic acid. Yield 8%, mp 164-168 °C (from
ethanol). Anal. Calcd. (C21H23NO7S (433.48)) C, H, N, S.
Methyl 5-chloro-3-[(2-methoxyphenyl)sulfonyl]-1H-indole-2-
carboxylate (18). MCPMA (0.16 g, 0.0009 mol) was added to an
ice-cold solution of 17 (0.13 g, 0.0004 mol) in chloroform (6 mL).
The reaction mixture was stirred at room temperature for 2 h, poured
on crushed ice, and extracted with chloroform. The organic solution
was shaken with a saturated solution of sodium hydrogen carbonate
and with brine and dried. After concentration to a small volume,
the solution was purified by silica gel column chromatography (ethyl
acetate-n-hexane 1:3 as eluent) to afford 18, yield 50%, mp 250
°C (from ethanol). Anal. Calcd. (C17H14ClNO5S (379.82)) C, H,
Cl, N, S.
Method C. Synthesis of Methyl 5-Chloro-3-[(3,5-dimethoxy-
phenyl)thio)]-1H-indole-2-carboxylate (25). 5-Chloro-1H-indole-
2-carboxylic acid (0.16 g, 0.0008 mol) was added by portions to a
mixture of sodium hydride (60% dispersion in mineral oil, 0.094
g, 0.0023 mol) in anhydrous DMF (4 mL) under a nitrogen stream
at 0 °C. After 15 min, 1,1′-(3,5-dimethoxyphenyl)disulfide (0.35
g, 0.001 mol) was added portionwise, then the reaction was heated
at 50 °C overnight under a nitrogen atmosphere. After cooling, the
mixture was poured onto crushed ice, made acidic with 2 N HCl
and extracted with ethyl acetate. The organic layer was separated,
washed with brine, and dried. Removal of the solvent gave a residue
that was triturated with cyclohexane and filtered to give 5-chloro-
3-[(3,5-dimethoxyphenyl)thio)]-1H-indole-2-carboxylic acid, which
was used as the crude product. The acid was dissolved in
dichloromethane (12 mL) and methanol (3 mL) and treated with
TMSDM (1.02 mL of a 2 N solution in hexane, 0.002 mol) while
stirring at room temperature for 30 min. After reduction to a small
volume, the suspension was treated with ethyl acetate (10 mL ×
5), and the solvent was evaporated. The crude product was purified
by silica gel column chromatography (ethyl acetate-n-hexane 1:5
as eluent) to give 25, yield 10%, mp 160-165 °C (from ethanol).
Anal. Calcd. (C18H16ClNO4S (377.84)) C, H, Cl, N, S.
Method D. General Procedure for the Synthesis of Com-
pounds 5-11, 37, and 40. Propyl 3-(Phenylthio)-1H-indole-2-
carboxylate (5). Methyl 1H-indole-2-carboxylate was obtained by
esterification of the corresponding acid using TMSDM, yield 88%,
mp 145-148 °C (from ethanol). Lit.,22 mp 149-151 °C (from ethyl
acetate-n-hexane). Methyl 3-(phenylthio)-1H-indole-2-carboxylate
was prepared by reaction of methyl 1H-indole-2-carboxylate with
N-(phenylthio)succinimide in the presence of boron trifluoride
diethyl etherate as above. The crude residue was purified by silica
gel column chromatography (ethyl acetate-n-hexane 1:3 as eluent),
yield 55%, mp 175-180 °C (from ethanol). Lit.,23 mp 178-180
°C (from diethyl ether-n-hexane). Lithium hydroxide monohydrate
(8.46 g, 0.20 mol) was added to a suspension of methyl 3-(phe-
nylthio)-1H-indole-2-carboxylate (18.52 g, 0.065 mol) in THF (250
mL) and water (250 mL). The mixture was stirred at room-
temperature overnight. After reduction to a small volume, it was
made acidic (pH ) 2) with 1 N HCl and extracted with ethyl acetate.
The organic layer was washed with brine, dried, and evaporated to
afford pure 3-(phenylthio)-1H-indole-2-carboxylic acid, yield 94%,
mp 161-162 °C (from glacial acetic acid). Lit.,23 160-162 °C
(from glacial acetic acid). To a cooled suspension of 3-(phenylthio)-
1H-indole-2-carboxylic acid (1.00 g, 0.0037 mol) in 1-propanol
(6.50 mL) was added dropwise thionyl chloride (0.33 mL) under
an anhydrous argon atmosphere. The reaction mixture was heated
at 70 °C for 5 h. After cooling, the suspension was filtered to afford
5, yield 52%, mp 132-135 °C (from ethanol). Anal. Calcd. (C18H17-
NO2S (311.40)) C, H, N, S.
Methyl 5-chloro-3-[(3-methoxyphenyl)sulfonyl]-1H-indole-2-
carboxylate (20) was synthesized as 18, starting from 19. Yield
92%, mp 170-173 °C (from ethanol). Anal. Calcd. (C17H14ClNO5S
(379.82)) C, H, Cl, N, S.
Methyl 5-chloro-3-[(4-methoxyphenyl)sulfonyl]-1H-indole-2-
carboxylate (22) was synthesized as 18, starting from 21. Yield
29%, mp 230-232 °C (from ethanol). Anal. Calcd. (C17H14ClNO5S
(379.82)) C, H, Cl, N, S.
Methyl 5-methoxy-3-(phenylsulfonyl)-1H-indole-2-carboxy-
late (30) was synthesized as 18, starting from 29. Yield 28%, mp
166-179 °C (from ethanol). Anal. Calcd. (C17H15NO5S (345.37))
C, H, N, S.
Ethyl 5,6-dimethoxy-3-(phenylsulfonyl)-1H-indole-2-carboxy-
late (39). Was synthesized as 18, starting from 38. Yield 47%, mp
264-273 °C (from ethanol). Anal. Calcd. (C19H19NO6S (389.42))
C, H, N, S.
N-(2-Methoxyphenylthio)succinimide. 2-Methoxythiophenol
(2.80 g, 0.02 mol) was added dropwise to an ice-cold mixture of
NCS (3.34 g, 0.025 mol) and anhydrous dichloromethane (30 mL)
under an argon atmosphere. After 1 h, additional NCS (0.4 g, 0.003
mol) was added, and the reaction mixture was stirred for 2.5 h.
Triethylamine (2.83 g, 3.9 mL, 0.028 mol) was added while stirring
for 15 min, and then dichloromethane (20 mL) and 1 N HCl (10
mL) were added. After shaking, the organic layer was dried,
concentrated to a small volume, and passed through a Celite column.
After evaporation of the solvent, the residue was triturated with
diethyl ether to give N-(2-methoxyphenylthio)succinimide, yield

Arylthioindoles Inhibitors of Tubulin Polymerization
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 953
60%, mp 165-168 °C, (from ethanol), Lit.,24 mp 173 °C. Anal.
Calcd. (C11H11NO3S (237.27)).
N-(3-Methoxyphenylthio)succinimide was synthesized as N-(2-
methoxyphenylthio)succinimide using 3-methoxythiophenol. Yield
60%, mp 147-149 °C, Lit.,25 mp 149-150 °C.
N-(4-Methoxyphenylthio)succinimide was synthesized as N-(2-
methoxyphenylthio)succinimide using 4-methoxythiophenol. Yield
60%, mp 135-140 °C, Lit.26 mp 100-106 °C. Anal. Calcd. (C11H11-
NO3S (237.27)).
washed with ice-cold Tris buffered saline containing 0.1% Tween
20 (TBST) (200 µL). A house vacuum source attached to an eight-
channel aspirator was used to remove the liquid from the plates.
Methanol (100 µL at -20 °C) was added to each well, and the
plate was placed at 4 °C for 10 min. The methanol was removed
by washing with TBST (2 × 200 µL). After further incubation at
r.t. for 2 h with SuperBlock (Pierce 37535) (200 µL), anti-Her-2
(c-erbB-2) antibody (Zymed Laboratories #28-004) (100 µL, 1:200
in SuperBlock) was placed in each well. The plate was incubated
overnight at 4 °C. For control wells, 1:200 dilution of a normal
rabbit IgG (Santa Cruz #SC-2027) in Superblock was used. Each
well was washed with TBST (2 × 200 µL) and incubated at r.t.
for 2 h with an anti-rabbit HRP-linked antibody (Sigma, A-0545)
(100 µL, 1:2000 in SuperBlock). Unreacted antibody was removed
by washing with TBST (3 × 200 µL), and the ECL Western blotting
reagent (Amersham #RPN2106) (100 µL) was added. The plate
was immediately read in an Analyst AD plate reader (Molecular
Devices). Each well was scanned for 0.1 s. Readings from wells
containing only control IgG and the corresponding HRP-linked
secondary antibody were set as background and subtracted from
all measured values. Luminescence readings from drug-treated cells
versus untreated cells were quantified and plotted against drug
concentration to give the EC50 values as the concentration of drug
that caused 50% decrease in luminescence.
Antimitotic Assay. Black, clear-bottom microtiter 96-well plates
(Corning Costar #3603) were used to accommodate experimental
cultures. MDA-MB-468 cells were seeded in each well at 8000
cells per well in growth medium (100 µL) and allowed to attach
overnight at 37 °C in a 5% CO2 atmosphere. Growth medium (100
µL) with drug or vehicle (dimethyl sulfoxide) was gently added to
the wells, and the plates were incubated at 37 °C in a 5% CO2
atmosphere for 24 h. Wells were washed with ice-cold TBST (2 ×
200 µL). A house vacuum source attached to an eight-channel
aspirator was used to remove the liquid from the 96-well plates.
Ice-cold methanol (100 µL) was added to each well, and the plate
was placed at 4 °C for 5 min. Methanol was removed by suction,
and plates were washed with ice-cold TBST (2 × 200 µL). Plates
were further incubated with SuperBlock blocking buffer (Pierce
#37535) (200 µL) for 2 h at r.t. The Tg-3 antibody (gift of Dr.
Davies, Albert Einstein College of Medicine) diluted 1:200 in
SuperBlock was placed in each well (100 µL), except the control
column that was treated with control antibody (Mouse IgM,
NeoMarkers, NC-1030-P). After 72 h, wells were washed with ice-
cold TBST (2 × 200 µL). The secondary antibody (goat Anti-Mouse
IgM, Southern Biotech #1020-05) was placed in each well at
1:2000 dilution in SuperBlock and incubated on a shaker at r.t. for
2 h. Unreacted antibody was removed by washing the plates with
ice-cold TBST (3 × 200 µL) for 5 min on a shaker. The ECL
Western Blotting Detection Reagents 1 and 2 in 1:1 mixture (100
µL) was placed in each well, and the plates were read immediately
in an Analyst AD plate reader (Molecular Devices). Luminescence
readings were imported into SOFTmax PRO 4.3.1. Antimitotic
activity was defined as a concentration dependent increase in
luminescence readings in compound-treated wells as compared to
vehicle-treated wells. The antibody (Tg-3), originally described as
a marker of Alzheimer’s disease, is highly specific for mitotic cells,
Tg-3 immunofluorescence being >50-fold more intense in mitotic
cells than in interphase cells.
1,1′-(3,5-Dimethoxyphenyl)disulfide. A mixture of 3,5-dimethoxy-
aniline (5.00 g, 0.033 mol), concentrated HCl (5.9 mL), and crushed
ice (8 g) was prepared and cooled to -5 °C. To this mixture was
added dropwise a solution of sodium nitrite (2.25 g, 0.033 mol) in
water (12 mL) over 45 min. The reaction mixture was stirred at 0
°C for an additional 15 min, at which time it was added to a solution
of potassium ethyl xantate (10.46 g, 0.065 mol) in water (31 mL)
at 65 °C. After the mixture was stirred for 30 min at 65 °C, it was
extracted with ethyl acetate. The organic layer was washed with
brine, dried, and evaporated to give 3,5-dimethoxybenzenethiol
(2.86 g, yield 51%), which was used without further purification.
A solution of iodine (6.10 g, 0.024 mol) in ethanol 96% (15 mL)
was added dropwise to a solution of 3,5-dimethoxybenzenethiol
(2.86 g, 0.017 mol) in the same solvent (5 mL) until a persistent
color was observed. After stirring for 10 min, the reaction mixture
was extracted with ethyl acetate. The organic layer was washed
with a saturated solution of sodium thiosulfate and with brine and
dried. Evaporation of the solvent gave 1,1′-(3,5-dimethoxyphenyl)-
disulfide, yield 52%, which was used without purification.
Biology. Tubulin Assembly. The reaction mixtures contained
0.8 M monosodium glutamate (pH 6.6 with HCl 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 controlled recording spectro-
photometer for 20 min at 30 °C. Extent of reaction was measured.
Full experimental details were previously reported.27
Colchicine Binding Assay. The reaction mixtures contained 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.28
MCF-7 Cell Growth. The above paper28 can also be referenced
for methodology of MCF-7 cell growth.
SCLC Cell Growth. Experiments were carried out following a
previously reported methodology.29
Hsp90 Competition Assay. Experiments were carried out as
previously reported.30 Briefly: Fluorescence polarization measure-
ments were performed on an Analyst AD instrument (Molecular
Devices, Sunnyvale, CA). Measurements were taken in black 96-
well microtiter plates (Corning #3650). The assay buffer contained
20 mM potassium HEPES pH 7.3, 50 mM KCl, 5 mM MgCl2, 20
mM Na2MoO4, and 0.01% NP40. Before each use, 0.1 mg/mL
bovine gamma globulin (Panvera Corporation, Madison, WI) and
2 mM dithiothreitol (Fisher Biotech, Fair Lawn, NJ) were added
to the buffer. GM-BODIPY (fluorescently labeled geldanamycin)
was synthesized as previously reported31 and was dissolved in
dimethyl sulfoxide to form a 10 µM solution. Recombinant Hsp90R
was purchased from Stressgen Bioreagents (cat. No. SPP-776),
(Victoria, BC, Canada). For the competition studies, each 96-well
contained 5 nM fluorescent GM-BODIPY, 30 nM Hsp90R and
potential inhibitor (initial stock in dimethyl sulfoxide) in a final
volume of 100 µL. The plate was left on a shaker at 4 °C for 24 h,
and the fluorescence polarization values in mP were recorded. EC50
values were determined as the competitor concentrations at which
50% of the fluorescent GM-BODIPY was displaced.
Molecular Modeling. All molecular modeling studies were
performed on a RM Innovator with Pentium IV 2.8 GHz processor,
running Linux Fedora Core 3 using Molecular Operating Environ-
ment (MOE) 2004.0317 and the FlexX module in SYBYL 7.0.16
The tubulin structure was downloaded from the PDB data bank
(http://www.rcsb.org/pdb/index.html PDB code: 1SA0).32 All the
minimizations were performed with MOE until RMSD gradient of
0.05 kcal mol-1 Å-1 was reached with the MMFF94x force field.
The partial charges were automatically calculated. Docking experi-
ments were carried out using the FlexX docking program of SYBYL
7.0 using the default settings. The output of FlexX docking was
visualized in MOE, and the scoring.svl script33 was used to identify
interaction types between ligand and protein. Molecular dynamics
Her2 Assay. The SKBr3 breast cancer cells were plated in black,
clear-bottom microtiter plates (Corning #3603) at 3000 cells/well
in growth medium (100 µL) and allowed to attach for 24 h at 37
°C and in a 5% CO2 atmosphere. Growth medium (100 µL) with
drug or vehicle (dimethyl sulfoxide) was carefully added to the
wells, and the plates were placed at 37 °C in the 5% CO2
atmosphere. Following a 24 h incubation with drugs, wells were

954 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
De Martino et al.
(14) De Martino, G.; La Regina, G.; Coluccia, A.; Edler, M. C.; Barbera,
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(15) Silvestri, R.; De Martino, G.; Artico, M.; Massa, S.; Marceddu, T.;
Loi, A. G.; Musiu, C.; La Colla, C. Indolyl Aryl Sulfones (IASs).
Part 1: SAR Studies and in vitro anti-HIV-1 activity against wt RT
and related mutants. J. Med. Chem. 2003, 46, 2482-2493.
(16) Chiosis, G.; Lucas, B.; Shtil, A.; Huezo, H.; Rosen, N. Development
of a purine-scaffold novel class of Hsp90 binders that inhibit the
proliferation of cancer cells and induce the degradation of Her2
tyrosine kinase. Bioorg. Med. Chem. 2002, 10, 3555-3564.
(17) Tripos SYBYL 7.0; Tripos Inc., 1699 South Hanley Rd, St. Louis,
MO 63144, USA. http://www.tripos.com.
was performed with MOE using the NVT environment for 600 ps
and constant temperature of 300 K using the MMFF94x force field
with a time step of 2 fs. Residues within 15 Å of the ligand were
allowed to move freely, keeping the rest of the protein fixed. The
binding site was soaked in a water sphere of 25 Å radius from the
sulfur atom of the ligand, and the total charge of the system included
in the water droplet did not require any adjustment. The water
molecules were energy minimized keeping the coordinates of the
protein-ligand complex fixed before the MD simulation. A distance
restraint of 25 Å with a weight of 100 between the oxygen atoms
of the water molecules and the sulfur atom of the ligand was also
applied.
(18) Molecular Operating Environment (MOE 2004.03). Chemical Com-
puting Group, Inc. Montreal, Quebec, Canada. http://www.chem-
comp.com.
(19) Bai, R.; Covell, D. G.; Pei, X. F.; Ewell, J. B.; Nguyen, N. Y.; Brossi,
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(20) For spectral data and elemental analysis of 13, Schlosser, K. M.;
Krasutsky, A. P.; Hamilton, H. W.; Reed, J. E.; Sexton, K. A highly
efficient procedure for 3-sulfenylation of indole-2-carboxylates. Org.
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Acknowledgment. The authors would like to thank all the
technical staff involved for their support in the realization of
this project.
Supporting Information Available: 1H NMR and IR spectral
data, and elemental analyses of new compounds 5-12, 14, 16-
20, 22, 25, 27, 28, 30-34, and 36-41 are available free of charge
via the Internet at http://pubs.acs.org.
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