Sulfonate derivatives of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone as highly active antimicrotubule agents. Synthesis, antiproliferative activity, and inhibition of tubulin polymerization

Article abstract of DOI:10.1021/jm0708984

Benzenesulfonate derivatives of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone were prepared and found to inhibit microtubule formation by an in vitro tubulin polymerization assay. Several analogues showed potent cytotoxic activity in an assay b

Full text of DOI:10.1021/jm0708984

J. Med. Chem. 2007, 50, 6059-6066
6059
Sulfonate Derivatives of Naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-Anthracenone as Highly
Active Antimicrotubule Agents. Synthesis, Antiproliferative Activity, and Inhibition of Tubulin
Polymerization
Anne Zuse,‡,# Peter Schmidt,§ Silke Baasner,§ Konrad J. Bo¨hm, Klaus Mu¨ller,# Matthias Gerlach,§ Eckhard G. Gu¨nther,§
Eberhard Unger, and Helge Prinz*,#
Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, CancerCare Manitoba, ON6010-675 McDermot AVenue,
Winnipeg, Canada, Institute of Pharmaceutical and Medicinal Chemistry, Westphalian Wilhelms-UniVersity, Hittorfstrasse 58-62,
D-48149 Mu¨nster, Germany, Aeterna Zentaris, Weismu¨llerstrasse 50, D-60314 Frankfurt, Germany, and Leibniz Institute for Age
Research-Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, D-07745 Jena, Germany
ReceiVed July 25, 2007
Benzenesulfonate derivatives of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone were prepared
and found to inhibit microtubule formation by an in vitro tubulin polymerization assay. Several analogues
showed potent cytotoxic activity in an assay based on K562 leukemia cells with IC50 values of <100 nM.
The methylamino analogue 14i was the most active compound in this assay (14i, IC50 K562: 0.05 µM).
Antiproliferative activities of selected compounds were additionally evaluated against a panel of 12 tumor
cell lines, including multi-drug-resistant phenotypes. All resistant cell lines were sensitive to these compounds.
Concentration-dependent flow cytometric studies showed that KB/HeLa cells treated with selected compounds
were arrested in the G2/M phases of the cell cycle. In competition experiments, these compounds strongly
displaced radiolabeled colchicine from its binding site in the tubulin, showing IC50 values lower than that
of colchicine. The results demonstrate that the antiproliferative activity is related to the inhibition of tubulin
polymerization.
Introduction
sources or by screening compound libraries in combination with
traditional medicinal chemistry.4,12
The mitotic spindle, constituted by microtubules generated
by polymerization of tubulin R-,â-dimers, remains an attractive
target for the development of compounds useful in anticancer
chemotherapy.1,2 A large number of antimitotic drugs displaying
a wide structural diversity have been identified to interfere with
tubulin.3,4 The Vinca alkaloids vinblastine (1, Chart 1) and
vincristine as well as the taxanes, such as taxol (INNa:
paclitaxel) and taxotere (INN: docetaxel) are widely used
clinically as important chemotherapeutic agents for the treatment
of many malignancies. It was mainly the discovery of these
compounds that has stimulated intense research aimed at
additional microtubule-targeting drugs. Colchicine (2) has
limited medicinal utility due to its narrow therapeutic window
but played an important role in the elucidation of the properties
and functions of tubulin and microtubules. Many natural
products, such as combretastatin A-45 (3) or the epothilones6
as well as some synthetic molecules including sulfonamide
E-70107 (4), thienopyrrolizinone8 (5), acridinyl-9-carboxamide
D-823189 (6), indolinone A-43241110 (7), or propenenitrile CC-
507911 (8) (Chart 1), to cite just a few, are known to mediate
cytotoxic activities through a binding interaction with tubulin.
In recent years, substantial efforts have been made to develop
small molecular colchicine-site binders derived from natural
We previously reported the potent in vitro antitumor activity
of 10-[(3-hydroxy-4-methoxybenzylidene)]-9(10H)-anthracenone
(10) and 9-[(4-hydroxy-3,5-dimethoxybenzylidene]naphtho[2,3-
b]thiophen-4(9H)-one (12, Chart 2).13,14 These compounds
strongly inhibited tumor cell growth as well as tubulin polym-
erization and caused significant arrest of mitosis. As part of
our search for novel antimitotic agents, we describe the synthesis
and biological evaluation of sulfonate derivatives related to
9(10H)-anthracenone (9) and naphtho[2,3-b]thiophen-4(9H)-one
(11) as tubulin polymerization inhibitors. Several compounds
strongly inhibited the growth of various tumor cell lines, acted
in a cell cycle dependent manner, and were found to be potent
inhibitors of tubulin polymerization. Antitubulin activities of
the most active compounds are comparable to those of the
reference compounds, such as nocodazole, podophyllotoxin, and
colchicine.
Chemistry
Sulfur-containing compounds, such as sulfonamide E-7010
(4),7 thienopyrrolizinones,8 and benzothiophene derivatives,8,15,16
proved effective inhibitors of tubulin polymerization. Previous
investigations by Gwaltney et al.17,18 have demonstrated the
potent antimitotic activities of sulfonate analogues of combre-
tastatin A4 and the sulfonamide E-7010 and have revealed that
the sulfonate can effectively replace the cis olefin of CA-4 (3).
These findings prompted us to synthesize sulfonates related to
9 and 11 in order to study their cytotoxic activity and their
inhibitory effect on tubulin polymerization. On the basis of our
previous chemical studies,13,14 it was expected that reaction of
9 and 11 with benzenesulfonyl chlorides would lead to sulfonate
derivatives. Keto-enol tautomerism of anthracenones as well
as in the thiophene analogues of anthrone has been described,19
and the formation of the sulfonates is suggested to be due to an
* To whom correspondence should be addressed. Tel: +49 251-8332195.
Fax: +49 251-8332144. E-mail: prinzh@uni-muenster.de.
‡ CancerCare Manitoba.
# Westphalian Wilhelms-University.
Leibniz Institute for Age Research-Fritz Lipmann Institute (FLI).
§ Aeterna Zentaris.
a Abbreviations: ADR, adriamycin; DMAP, 4-dimethylaminopyridine;
G, Gap; M, mitosis; INN, international nonproprietary name; ITP, inhibition
of tubulin polymerization; Kip, kinase inhibitor protein; MDR, multi-drug-
resistant; MTP, microtubule protein; ND, not defined; TBAB, tetra-n-
butylammonium bromide; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-
2H-tetrazolium-5-carboxanilide; VCR, vincristine.
10.1021/jm0708984 CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/31/2007

6060 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Chart 1. Examples of Tubulin Interacting Agents
Zuse et al.
Chart 2. Structure of 9, 11, and Related Benzylidene Derivatives 10 and 12
O-sulfonylation of the enol tautomeric form of the starting 9
and 11 under basic conditions. The general method for the
synthesis of the sulfonates is outlined in Scheme 1. Most of
the benzenesulfonyl chlorides as well as 9 were commercially
available, and 11 was prepared as previously described.14
Additional modifications concerned substitution in the tricyclic
ring system. Accordingly, 1,8-dichloro- and 4,5-dichloro-
9(10H)-anthracenone were prepared as described.20,21 The
desired sulfonates 13a-c, 14a-g, 14l, 17, and 18 were obtained
by a sulfonylation reaction of 9, 11, 15, and 16 with ap-
propriately substituted benzenesulfonyl chlorides under standard
conditions in the presence of sodium hydroxide or pyridine/
DMAP in the case of 11. Interestingly, as a side product of the
sulfonylation reaction of 9, we isolated the chloro-substituted
compound 13b. Reduction of the nitro group of 14g provided
the amino compound 14h, which was subsequently N-mono-
and bimethylated to obtain target compounds 14i and 14j
(Scheme 2). The 4-hydroxyaminobenzensulfonic acid derivative
14k was prepared using a Pd/C-catalyzed hydrogenation of
14g.22-24 Furthermore, we explored the effect of bioisosteric
replacement of the 10-carbon atom in 9 with nitrogen. To this
end, the O-sulfonylation of commercially available 9(10H)-
acridone (19) with 4-methoxybenzenesulfonyl chloride and
NaOH in the presence of tetra-n-butylammonium bromide
(TBAB) as a phase transfer catalyst25 was studied. However,
no satisfactory analytical data were obtained for acridone-
derived 21, since traces of 19 could not be separated. By
contrast, 1,2,3,4-tetrahydro-9-acridanone (20) smoothly yielded
sulfonate 22 (Scheme 3) under identical conditions.
Biological Results and Discussion
In Vitro Cell Growth Inhibition Assay. In an initial screen,
the compounds were evaluated for antiproliferative activity
against the human chronic myelogenous leukemia cell line
K56226 which is widely used for potential antitumor compounds.
Cell proliferation was determined directly by counting the cells
with a hemocytometer after 48 h of treatment. The structures
and the growth inhibitory data of the synthesized sulfonates are
listed in Table 1. With the exception of the nitro compounds
14g and 14l most compounds showed IC50 values in the sub-
micromolar (lower than 1 µM) range. In the benzenesulfonic
acid naphtho[2,3-b]thiophen-4-yl-ester series, three compounds
(14b, 14i, and 14j) displayed strong antiproliferative activity
with IC50 values of lower than 0.1 µM. A similar activity was
found for the 4-methoxybenzenesulfonic acid anthracen-9-yl
ester (13a) (IC50 K562: 0.09 µM). Replacement of the 4-meth-
oxy group of 14b (IC50 K562: 0.06 µM) with a 4-methyl group
(14e) led to a loss of potency. A similar loss of potency was
observed for the isomeric 14c versus 14f, respectively. In
addition, our findings indicate that a methoxy group located at
the para-position to the sulfonyl part resulted in strong anti-
proliferative activity, while shifting it to the meta-position
decreased the activity (13a versus 13c, 14b versus 14c).
Noteworthy, potent inhibition of tubulin polymerization was
found for 13c and 14c (vide infra). Chloro-substituted compound

Antimicrotubule Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6061
Scheme 1a,b
against a panel of five tumor cell lines derived from solid tumors
was measured by cellular metabolic activity using the XTT (2,3-
bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxa-
nilide) assay.27 Generally, compound 14b was found to be
slightly more potent than 13a and showed activities with IC50
values in the range of 0.2 µM toward several proliferating cell
lines (Table 2). Both compounds provide a good example of
the classical bioisosteric equivalence between benzene and
thiophene, with both compounds exhibiting nearly the same
biological activity. However, the thiophene derivative 14b was
found to be three-fold more active against the NCI-H460 cell
line than 13a. Interestingly, 13a and 14b were not active against
RKO cells (human colon adenocarcinoma) with ectopic induc-
ible expression of cyclin-dependent kinase inhibitor p27kip1 28
.
By contrast, growth of proliferating RKO cells was strongly
inhibited by 13a and 14b with IC50 values of 0.28 and 0.18
µM, respectively, indicating activity toward cycling cells. A
major obstacle to the treatment of many human cancers is the
development of multiple drug resistance (MDR) in patients and
a loss of efficacy over time, meaning that cancer cells do not
respond to treatment or develop a broad spectrum resistance to
several anticancer drugs, among them antitubulin agents.29,30
Multiple drug resistance is mediated, among other factors, by
overexpression of transmembrane cellular pumps, such as the
170 kDa P-glycoprotein (pgp),31 encoded by the mdr1 gene and
the 180 kDa MDR protein (MRP).32 Important aspects concern-
ing the key mechanisms of antimicrotubule drug resistance have
recently been reviewed.33 The antiproliferative activity of 13a
and 14b against tumor cell lines with different resistance
phenotypes was evaluated in an XTT-based assay. On the whole,
as documented by the IC50 data (Table 3), 13a and 14b were
effective against the cell lines tested and retained activity in
cell lines with various multiple drug resistance phenotypes. This
feature was distinct from paclitaxel and vindesine because
LT12MDR, L1210VCR, and P388ADR cell lines were more
resistant to these chemotherapeutics than the nonresistant cell-
lines. Therefore, the sulfonate analogues 13a and 14b are
inhibitors of tumor cell proliferation and are not subject to
resistance mediated by drug efflux activity by overexpression
of pgp170.
a R1-R5 are defined in Table 1. bReagents and conditions: (a) 9(10H)-
anthracenone, 2 N NaOH/THF, rt; (b) naphtho[2,3-b]thiophen-4(9H)-one,
CH2Cl2, DMAP, pyridine, N2, 0-5 °C to rt; (c) 4-(MeO)PhSO2Cl, 2 N
NaOH/THF, rt.
Effect on Cell-Cycle Progression. To gain further insight
into the mode of action, representative compounds 13a and 14b
were assayed for their effects on cell cycle using an established
KB/HeLa (human cervical epitheloid carcinoma) cell-based
assay system. For a thorough comparison of 13a and 14b with
known G2/M cell cycle inhibitors, subconfluent KB/HeLa cells
were exposed to test compounds, and the percentage of cells in
G2/M phase after 24 h was plotted against different concentra-
tions of the compounds. The concentration for 50% cells arrested
in G2/M phase by 13a and 14b was found to be in the range of
0.13 µM (Table 4), thus being somewhat less active than
nocodazole (EC50 0.09 µM). For comparison, the recently
described anthracenone derivative 10 had an EC50 value of 0.2
µM in this assay. In summary, the effect of compounds 13a
and 14b on cell cycle progression correlated well with their
strong antiproliferative and antitubulin activities and is similar
to that observed for the majority of antimitotic agents. However,
the reference compounds were more potent than any of the new
compounds examined in this assay.
13b was determined to be clearly less potent than 13a (13a,
0.09 µM vs 0.35 µM for 13b). The replacement of the methoxy
group of 14b with a dimethylamino (14j) or a methylamino
(14i) group in the benzenesulfonyl part retained strong activity
with IC50 values in the range of 0.05-0.08 µM, indicating that
the methoxy and the methylamino groups are obviously
bioequivalent at the C4-position of the benzenesulfonyl part.
Activities were comparable to that of 13a which is a bioisostere
of 14b. The methylamino analogue 14i was the most active
compound in this assay (IC50 K562: 0.05 µM), being slightly
less potent than adriamycin (IC50 K562: 0.01 µM) and
colchicine (IC50 K562 0.02 µM). The amino-substituted deriva-
tive 14h had an IC50 of 0.13 µM, thus being slightly less active
than its methylated congeners 14i and 14j. However, a
substantial loss of activity was found for the 4-hydroxyamino-
substituted compound 14k. The 1,8- and 4,5-dichloro-9(10H)-
anthracenone-derived compounds 17 and 18 showed only
moderate cytotoxic effects. Moreover, naphtho[2,3-b]thiophen-
4(9H)-one (11) displayed only weak activity (data not shown),
documenting that the antiproliferative activity is closely related
to the sulfonate structure.
In Vitro Tubulin Polymerization Assays. To investigate
whether the antiproliferative activities were related to an
interaction with tubulin, 15 analogues were assayed for their
inhibitory effects on tubulin polymerization using assay condi-
tions as described previously.14 Depending on the temperature-
Effect on Growth of Different Tumor Cell Lines. To further
characterize the cytotoxicity profile of these compounds, the
effect of the highly active methoxy analogues 13a and 14b

6062 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Zuse et al.
Scheme 2a
a Reagents and conditions: (a) SnCl2·2H2O, C2H5OH, N2, reflux, 18 h; (b) (CH3O)2SO2, acetone, K2CO3, 50 °C, 24 h; (c) CH3I, DMF, K2CO3, 80 °C,
24 h; (d) Pd/C, C2H5OH, H2, rt, 6 h.
Scheme 3a
the tumor cell growth assay and the tubulin polymerization assay
differ in a number of further important issues, such as tubulin
concentration present within the cells and during microtubule
formation outside a cell, the presence of different types of
microtubule-associated proteins, and the possible effects of
regulatory proteins expressed in the cell but being absent in
the tubulin assay. The 4-methoxy-substituted compound 13a
(IC50 1.20 µM) was as good an inhibitor of tubulin polymeri-
zation as colchicine. Furthermore, compounds 14b, 14i, and 14j
proved to be exceptionally strong inhibitors of tubulin polym-
erization (IC50 e 1.0 µM), as did the reference drugs with the
exception of colchicine (1.4 µM). For these four compounds,
the order of inhibitory action on tubulin polymerization was
14i > 14b > 14j > 13a, which was consistent with the results
from the K562 cell growth inhibition assay. Again, this finding
indicates that a methoxy group plays an essential role to exhibit
strong antitubulin and antiproliferative activities within this
series of compounds. The nitro compound 14g did not show
any appreciable activity as an inhibitor of tubulin polymeriza-
tion. The introduction of chloro substituents into the anthra-
cenoid core caused a complete loss of activity as seen with 17
and 18, suggesting that electronic or lipophilic factors account
for the loss of activity observed with these two compounds.
Moreover, compound 22 proved inactive as an inhibitor of
tubulin polymerization, indicating that steric factors such as
planarity of the tricyclic ring system may contribute to a potent
tubulin inhibitory activity. Moderate inhibitors of tumor cell
growth showed only poor inhibitory effects on tubulin polym-
erization (IC50 values ranging from 2.8 to >10 µM).
Microtubule-destabilizing drugs often reveal specific tubulin
binding sites. Therefore, we assessed the capability of 13a and
14b to compete with either colchicine or paclitaxel for binding
to tubulin using a competitive scintillation proximity assay.34
Both 13a and 14b competitively inhibited [3H]colchicine binding
to biotinylated tubulin (Figure 2) with exceptionally low IC50
values of 0.56 µM (13a) and 0.51 µM (14b) as compared to
colchicine (1.05 µM). The results were consistent with their
growth inhibitory and tubulin polymerization inhibitory activi-
ties. However, the compounds did not compete with [3H]-
paclitaxel. No stabilization of the colchicine binding was
observed, as it is documented for Vinca site binders.35,36
Therefore, we conclude that binding to tubulin at the colchicine-
binding site is highly probable and that the capacity to interact
with the mitotic spindle contributes to the antiproliferative
activity of the compounds.
a Reagents and conditions: (a) 4-(MeO)PhSO2Cl, NaOH 50%, THF/
H2O, TBAB.
dependent equilibrium between R/â-tubulin dimers and micro-
tubules, physiological temperature (37 °C) favors polymerization
of tubulin dimers into microtubules in the presence of GTP and
some cofactors, which is detected by an increase in turbidity.
Depolymerization is induced by shifting the temperature to
2 °C. If a tubulin polymerization inhibitor binds to or interferes
with tubulin, the assembly steady-state level is decreased, as
exemplified for 13a (Figure 1). The results obtained with the
test agents are summarized in Table 1. For comparison, the data
of the potent antimitotic compounds colchicine, podophyllotoxin,
nocodazole, and vinblastine are also presented. With the
exception of compounds 14a, 14g, 17, 18, and 22, all com-
pounds tested were found to inhibit tubulin polymerization.
When comparing the inhibition of tubulin polymerization versus
the growth inhibitory effect, we found a good correlation for
most of the active compounds, but not for all of them. A
noticeable finding is the high potency of compound 13c as an
inhibitor of tubulin polymerization (IC50 0.39 µM). A similar
observation was found for 14c, possibly indicating the signifi-
cance of a 3-methoxy group, which is a well-defined pharma-
cophore for the inhibition of tubulin polymerization.9 The strong
decrease in potency on K562 cells in the case of 13c and 14c
can possibly be rationalized by a limited penetration into the
cell or any other mechanism limiting the accessibility of these
molecules to the cellular tubulin. It has to be considered that

Antimicrotubule Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6063
Table 1. Antiproliferative Activity of 13a-c, 14a-m, 17, 18, and 22 against K562 Cells and Antitubulin Activities
compd
R1
R2
R3
R4
R5
K562 IC50a [µM]
ITP IC50b [µM]
13a
13b
13c
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
17
H
H
OCH3
H
OCH3
OCH3
H
H
OCH3
H
OCH3
CH3
H
NO2
NH2
NHCH3
N(CH3)2
NHOH
NO2
NH2
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
0.09
0.35
0.48
0.80
0.06
0.25
0.17
0.44
0.53
2.3
0.13
0.05
0.08
0.40
1.4
0.28
10
1.7
0.83
0.02
ND
ND
0.001
0.01
1.20
0.39
>14
0.91
0.29
1.27
1.05
0.74
>14
0.71
0.29
0.94
ND
H
OCH3
OCH3
H
CH3
H
H
H
H
H
H
H
Cl
H
OCH3
OCH3
H
ND
ND
>10
>10
>10
1.4
0.76
0.35
0.13
ND
18
22
H
Cl
colchicine
nocodazole
podophyllotoxin
vinblastine sulfate
adriamycin
a IC50, concentration of drug required for 50% inhibition of cell growth (K562). Cells were treated with drugs for 2 days. IC50 values are the means of
at least three independent determinations (SD < 10%). b ITP ) inhibition of tubulin polymerization; IC50 values were determined after 20 min at 37 °C and
represent the concentration for 50% inhibition of the maximum tubulin assembly rate.
Table 2. Cytotoxic Activity of 13a and 14b against Different Tumor Cell Lines
IC50 [µM]a
RKOp27kip1
(human colon adenocarcinoma)
KB/HeLa
(cervix)
SKOV3
(ovary)
SF268
(glioma)
NCI-H460
(lung)
compd
13a
not induced
induced
0.37
0.18
0.01
0.14
0.03
0.25
0.21
0.01
0.17
0.05
0.34
0.26
0.01
0.30
0.05
0.64
0.21
0.01
0.15
0.07
0.28
0.18
0.01
0.11
0.02
>9
>9
14b
paclitaxel
nocodazole
colchicine
>3
>14
>14
a IC50 values were determined from XTT proliferation assays after incubation with test compound for 48 h. All experiments were performed at least in
two replicates (n ) 2), and IC50 data were calculated from dose-response curves by nonlinear regression analysis.
Conclusion
arrest was demonstrated for these compounds. Notably, no
growth inhibitory effect was found in cell cycle arrested cells.
Whereas the effectiveness of numerous clinically useful drugs
is limited by the fact that they are substrates for the efflux pumps
Pgp170 and MRP, compounds 13a and 14b were found to be
active toward parental tumor cell lines and multi-drug-resistant
cell lines, with 14b being slightly more potent. This feature is
distinct from those of paclitaxel and vindesine, because three
cell lines were more resistant to these agents than the nonre-
sistant cell lines. As pointed out, it has previously been shown
that the sulfonate group is an effective replacement for the cis
olefin of CA-4.17 Therefore, in the figurative sense, the
sulfonates described herein can be considered as anthracenoid
analogues of combretastatin A4. As low aqueous solubilities
and short iv half-lives have been documented for sulfonate
A novel series of sulfonate derivatives of 9(10H)-anthra-
cenone and naphtho[2,3-b]thiophen-4(9H)-one was synthesized
and we have identified several representatives of this series to
be highly potent antiproliferative agents and inhibitors of tubulin
polymerization. Similar to many other inhibitors of tubulin
polymerization, selected compounds 13a and 14b were effica-
cious in inhibiting tumor cell proliferation with IC50 values at
the nanomolar level. Our studies revealed that a 4-methoxy or
a 4-methylamino substitution pattern in the terminal phenyl ring
is critical for strong inhibition of tumor cell proliferation and
inhibition of tubulin polymerization. Sulfonates 13a and 14b
are potent inhibitors of the colchicine binding to tubulin and
interact most likely with tubulin at the colchicine site. As with
other microtubule-interacting agents, the induction of G2/M

6064 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Zuse et al.
Table 3. Antiproliferative Activity of 13a and 14b, Paclitaxel, Nocodazole, and Vindesine against Tumor Cell Lines with Different Resistance
Phenotypes (XTT Assay)
IC50 [µM]a
LT12
(blood)
LT12 MDR
(blood)
L1210
(blood)
L1210 VCR-resistant
(blood)
P388
(blood)
P388 ADR-resistant
(blood)
compd
13a
0.44
0.17
0.006
0.30
0.001
0.36
0.19
0.40
0.05
0.26
0.54
0.19
0.06
0.06
0.02
0.46
0.20
>5
0.07
>5
0.38
0.18
0.04
0.07
0.01
0.27
0.18
>5
0.05
1.14
14b
paclitaxel
nocodazole
vindesine
a IC50 values were determined from XTT proliferation assays after incubation with test compound for 48 h. All experiments were performed at least in
two replicates (n ) 2), and IC50 data were calculated from dose-response curves by nonlinear regression analysis.
Table 4. Cell Cycle Analysis of KB/HeLa Cells Treated with 13a and
14b and Reference Compounds Vincristine, Colchicine, Paclitaxel, and
Nocodazole
antitumor activities, we believe that compounds of this structural
class are attractive for further structural modifications and that
our findings may provide useful information for the design of
novel antitumor agents. Investigations on the role of the
anthracene and naphthothiophene moiety for antimitotic activity
are in progress, and results from other modifications will be
reported in due course.
13a 14b colchicine nocodazole paclitaxel vincristine
EC50 [nM]a 135 125
14
91
49
2.4
a EC50 values were determined from dose-response cell cycle analysis
experiments and represent the concentration for 50% cells arrested in G2/M
phase after 24 h. All experiments were performed at least in two replicates
(n ) 2), and EC50 data were calculated from dose-response curves by
nonlinear regression analysis (GraphPad Prism).
Experimental Section
Melting points were determined with a Kofler melting point
apparatus and are uncorrected. Spectra were obtained as follows:
1H NMR spectra were recorded with a Varian Mercury 400 plus
(400 MHz) spectrometer, using tetramethylsilane as an internal
standard. Fourier-transform IR spectra were recorded on a Bio-
Rad laboratories Typ FTS 135 spectrometer and analysis was
performed with WIN-IR Foundation software. Elemental analyses
were performed at the Mu¨nster microanalysis laboratory, using a
vario EL III CHNOS elemental analyzer (Elementar Analysensys-
teme GmbH), and all values were within (0.4% of the calculated
composition. Mass spectra were recorded in the EI mode using a
MAT GCQ Finnigan instrument. All organic solvents were ap-
propriately dried or purified prior to use. Benzenesulfonyl chlorides
were obtained from commercial sources. Analytical TLC was done
on Merck silica 60 F254 alumina coated plates (E. Merck, Darms-
tadt). Silica gel column chromatography was performed using Acros
1
60-200 mesh silica gel. All new compounds displayed H NMR,
FTIR, and MS spectra consistent with the assigned structure. Yields
have not been optimized. Chromatography solvent (vol %): EE )
ethyl acetate; H ) hexane; M ) methanol; MC ) methylene
chloride. Elemental analyses were within (0.4% of calculated
values, except where stated otherwise.
4-Methoxybenzenesulfonic Acid Anthracen-9-yl Ester (13a).
9(10H)-anthracenone (9) (0.97 g, 5 mmol) and 4-methoxybenze-
nesulfonyl chloride (1.03 g, 5 mmol) were dissolved in a mixture
of 2 N NaOH (20 mL) and THF (10 mL) at room temperature
under N2. The mixture became orange and was thereafter stirred
until the reaction was complete (TLC control). Then, the reaction
mixture was poured into water (250 mL) and HCl (50 mL) and
extracted with CH2Cl2 (3 × 75 mL). The organic phase was dried
over Na2SO4, washed with water, and then evaporated. Purification
by silica gel chromatography (MC) afforded 13a as a pale-yellow
powder (0.37 g, 20% yield): mp 162-163 °C.
Figure 1. Inhibition of in vitro tubulin polymerization (microtubule
formation) at 37 °C by various concentrations of 13a. Turbidity was
recorded at 360 nm (MTP 1 mg/mL). Tubulin assembly in the absence
of inhibitor gave the control readings. IC50 values were determined
after 20 min and represent the concentration for 50% inhibition of the
maximum tubulin polymerization level.
4-Methoxybenzenesulfonic Acid 10-Chloroanthracen-9-yl es-
ter (13b). The title compound was obtained from (9) (0.97 g, 5
mmol) and 4-methoxybenzenesulfonyl chloride (1.03 g, 5 mmol),
as described for 13a. Purification by silica gel chromatography
(MC) afforded 13b as a yellow crystalline powder (54 mg, 3%
yield): mp 158-159 °C.
3-Methoxybenzenesulfonic Acid Anthracen-9-yl ester (13c).
The title compound was obtained from (9) (0.97 g, 5 mmol) and
3-methoxybenzenesulfonyl chloride (1.03 g, 5 mmol), as described
for 13a. Purification by silica gel chromatography (MC/H, 8:2)
afforded 13c as fine yellow crystals (0.13 g, 7% yield): mp 130
°C.
Figure 2. [3H]Colchicine competition binding assay of 13a, 14b, and
colchicine. Radiolabeled colchicine, unlabeled compound, and biotin-
labeled tubulin were incubated together for 2 h at 37 °C.
analogues of CA-4, resulting in a poor pharmacokinetic
behavior, this may also account for the sulfonates presented
herein. The pharmacokinetic profile of our compounds has not
been studied yet. Nevertheless, due to their attractive in vitro
Benzenesulfonic Acid Naphtho[2,3-b]thiophen-4-yl Ester (14a).
According to a literature method,17,37 to a solution of naphtho[2,3-

Antimicrotubule Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6065
b]thiophen-4-one (11) (0.7 g, 3.5 mmol) in absolute CH2Cl2 (20
mL) were added DMAP (0.15 g) and pyridine (5 mL) under N2 at
0 °C. Then, a solution of benzenesulfonyl chloride (0.62 g, 0.45
mL, 3.5 mmol) in absolute CH2Cl2 was added dropwise. The
mixture was stirred until the reaction was complete (TLC control).
Thereafter, the reaction mixture was poured into water (250 mL)
and extracted with CH2Cl2 (3 × 75 mL). The organic phase was
dried over Na2SO4, washed with water, and then evaporated.
Purification by silica gel chromatography (MC/H 1:1) afforded 14a
as white crystals (0.49 g, 38% yield): mp 152-154 °C.
4-Methoxybenzenesulfonic Acid Naphtho[2,3-b]thiophen-4-
yl Ester (14b). The title compound was prepared from 11 (0.5 g,
2.5 mmol) and 4-methoxybenzenesulfonyl chloride (0.52 g, 2.5
mmol) as described for 14a. Purification by silica gel chromatog-
raphy (MC/H 8:2) afforded 14b as fine, white crystals (0.44 g, 47%
yield): mp 155-156 °C.
3-Methoxybenzenesulfonic Acid Naphtho[2,3-b]thiophen-4-
yl Ester (14c). The title compound was prepared from 11 (0.8 g,
4 mmol) and 3-methoxybenzenesulfonyl chloride (0.82 g, 0.56 mL,
4 mmol) as described for 14a. Purification by silica gel chroma-
tography (MC/H 8:2) afforded 14c as a white powder (0.55 g,
38%): mp 110-112 °C.
3,4-Dimethoxybenzenesulfonic Acid Naphtho[2,3-b]thiophen-
4-yl Ester (14d). The title compound was prepared from 11 (0.85
g, 4.23 mmol) and 3,4-dimethoxybenzenesulfonyl chloride (1 g,
4.23 mmol) as described for 14a. Purification by silica gel
chromatography (H/PE 1:1) afforded 14d as white crystals (0.43
g, 25%) yield): mp 163-165 °C.
4-Methylbenzensulfonic Acid Naphtho[2,3-b]thiophen-4-yl
Ester (14e). The title compound was prepared from 11 (0.6 g, 3
mmol) and 4-methylbenzenesulfonyl chloride (0.57 g, 3 mmol) as
described for 14a. Purification by silica gel chromatography (MC/H
1:1) afforded 14e as a fine white powder (0.47 g, 44%) yield): mp
164-165 °C.
3-Methylbenzenesulfonic Acid Naphtho[2,3-b]thiophen-4-yl
Ester (14f). The title compound was prepared from 11 (0.8 g, 4
mmol) and 3-methylbenzenesulfonyl chloride (0.76 g, 0.57 mL, 4
mmol) as described for 14a. Purification by silica gel chromatog-
raphy (MC/H 1:1) afforded 14f as colorless crystals (0.64 g,
45%): mp 122-124 °C.
4-Nitrobenzenesulfonic Acid Naphtho[2,3-b]thiophen-4-yl Es-
ter (14g). The title compound was prepared from 11 (0.8 g, 4 mmol)
and 4-nitrobenzenesulfonyl chloride (0.89 g, 4 mmol) as described
for 14a. Purification by silica gel chromatography (MC/H 1:1)
afforded 14g as yellow crystals (0.14 g, 9%) yield): mp 199-201
°C.
4-Aminobenzenesulfonic Acid Naphtho[2,3-b]thiophen-4-yl
Ester (14h).38 14g (0.39 g, 1 mmol) was suspended in absolute
ethanol (50 mL). Then, SnCl2‚2H2O (3.89 mmol) was added, and
the mixture was refluxed for 18 h under N2 until the reaction was
complete. Thereafter, the mixture was cooled and the solvent
evaporated. Purification by silica gel chromatography (MC) afforded
14h as a fine white powder (0.27 g, 76% yield): mp 187-189 °C.
4-Methylaminobenzensulfonic Acid Naphtho[2,3-b]thiophen-
4-yl Ester (14i). 14h (0.15 g, 0.42 mmol) and dry K2CO3 (0.58 g,
4.2 mmol) were suspended in absolute acetone (50 mL) under N2.
Dimethyl sulfate (0.39 g, 0.3 mL, 3.15 mmol) was added dropwise,
and the mixture was heated at 50 °C under nitrogen until the
reaction was completed (TLC control). The reaction mixture was
then cooled and filtered with suction. The filtrate was then poured
into water (200 mL) and extracted with CH2Cl2 (4 × 25 mL). The
combined CH2Cl2 extracts were washed, dried over Na2SO4, and
then evaporated. The residue was purified by chromatography
(MC/H 8:2) and afforded 14i as white crystals (0.03 g, 20%): mp
178-180 °C.
filtered with suction. The filtrate was then poured into water (100
mL) and extracted with CH2Cl2 (4 × 30 mL). The combined organic
extracts were washed with water (3 × 25 mL), dried over Na2SO4,
and then evaporated. Purification by silica gel chromatography
(MC/H 8:2) afforded 14j as white crystals (0.05 g, 43% yield):
mp 221-224 °C.
4-Hydroxyaminobenzensulfonic Acid Naphtho[2,3-b]thiophen-
4-yl Ester (14k). According to literature methods,22-24 14g (0.8 g,
2.10 mmol) was suspended in absolute ethanol (50 mL) and
hydrogenated over 10% Pd/C (0.5 g) at room temperature and
atmospheric pressure. The mixture was stirred until the reaction
was complete. Then, the catalyst was removed by filtration, and
the solution was evaporated. Purification by silica gel chromatog-
raphy (MC) afforded 14k as a pale-yellow powder (0.39 g, 38%):
mp 168-171 °C.
2-Methoxy-4-nitrobenzenesulfonic Acid Naphtho[2,3-b]-
thiophen-4-yl Ester (14l). The title compound was obtained from
11 (0.8 g, 4 mmol) and 2-methoxy-4-nitrobenzenesulfonyl chloride
(1 g, 4 mmol) as described for 14a. Purification by silica gel
chromatography (MC/H 1:1) afforded 14l as yellow crystals (0.61
g, 37% yield): mp 171-173 °C.
4-Amino-2-methoxybenzenesulfonic Acid Naphtho[2,3-b]-
thiophen-4-yl Ester (14m). The title compound was obtained from
14l (0.5 g, 1.2 mmol) as described for 14h.
Purification by silica gel chromatography (MC/H 8:2) afforded
14m as a white powder (0.04 g, 8% yield): mp 218-220 °C.
4-Methoxybenzenesulfonic Acid 1,8-Dichloroanthracen-9-yl
Ester (17). The title compound was obtained from 1,8-dichloro-
9(10H)-anthracenone (15, 1.32 g, 5 mmol) and 4-methoxybenze-
nesulfonyl chloride (1.03 g, 5 mmol) as described for 13a.
Purification by silica gel chromatography (MC) afforded 17 as fine
yellow crystals (0.79 g, 37% yield): mp 177 °C.
4-Methoxybenzenesulfonic Acid 4,5-Dichloroanthracen-9-yl
Ester (18). The title compound was obtained from 4,5-dichloro-
9(10H)-anthracenone (16, 1.32 g, 5 mmol) and 4-methoxybenze-
nesulfonyl chloride (1.03 g, 5 mmol) as described for 13a.
Purification by silica gel chromatography (MC) afforded 18 as fine
yellow crystals (0.44 g, 20% yield): mp 176-177 °C.
4-Methoxybenzenesulfonic Acid 1,2,3,4-Tetrahydroacridin-
9-yl Ester (22). A suspension of 1,2,3,4-tetrahydro-9-acridanone
(20, 1.25 g, 6.25 mmol), TBAB (0.3 g, 1.03 mmol), and 50%
sodium hydroxide (10 mL) in THF (20 mL) and water (5 mL) was
stirred vigorously for 20 min. A solution of 4-methoxybenzene-
sulfonyl chloride (2.58 g, 12.5 mmol) in THF 5 mL was added
dropwise to the reaction mixture. The mixture was stirred until the
reaction was complete (TLC control), poured into water (250 mL)
and HCl (50 mL), and extracted with EtOAc (3 × 50 mL). The
combined organic layer was dried over anhydrous Na2SO4, washed
with water, and then concentrated under vacuum.
Purification by silica gel chromatography (MC/MeOH, 9.97:0.03)
afforded 22 as white crystalline powder (1.44 g, 62% yield): mp
172-173 °C.
Biological Assay Methods. These were described previously in
full detail.14
Acknowledgment. We wish to thank Cornelia Lang, Britta
Kroenert, Sonja Seidel, Katrin Zimmermann, Magnus Bro¨cker,
and Angelika Zinner for excellent technical assistance.
Supporting Information Available: IR, 1H NMR, and MS data
of new compounds 13a-c, 14a-m, 17, 18, and 22; cell cycle
analysis of KB/HeLa cells treated with 13a, 14b, and reference
compounds; table of elemental analysis results of all target
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
4-Dimethylaminobenzensulfonic Acid Naphtho[2,3-b]thiophen-
4-yl Ester (14j). Following a literature method,39,40 14h (0.11 g,
0.31 mmol) and dry K2CO3 (0.09 g, 0.62 mmol) were suspended
in absolute DMF (20 mL) under N2. Methyl iodide (1.6 g, 0.7 mL,
0.01 mol) was added at room temperature, and the mixture was
heated at 80 °C. After 24 h the reaction mixture was cooled and
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