Novel 2-Substituted quinolin-4-yl-benzenesulfonate derivatives: Synthesis, antiproliferative activity, and inhibition of cellular tubulin polymerization

Article abstract of DOI:10.1002/cmdc.201100121

A series of 2-substituted quinolin-4-yl-benzenesulfonate derivatives were synthesized for the purpose of evaluating antiproliferative activity. Structure-activity relationships of the newly synthesized compounds against human lymphoblastic leukemia and va

Full text of DOI:10.1002/cmdc.201100121

DOI: 10.1002/cmdc.201100121
Novel 2-Substituted Quinolin-4-yl-benzenesulfonate
Derivatives: Synthesis, Antiproliferative Activity, and
Inhibition of Cellular Tubulin Polymerization
Rajesh Kakadiya,[a] Yi-Chen Wu,[b] Huajin Dong,[c] Hsiao-Hui Kuo,[b] Ling-Huei Yih,*[b]
Ting-Chao Chou,[c] and Tsann-Long Su*[a, d]
A series of 2-substituted quinolin-4-yl-benzenesulfonate deriva-
tives were synthesized for the purpose of evaluating antiproli-
ferative activity. Structure–activity relationships of the newly
synthesized compounds against human lymphoblastic leuke-
mia and various solid tumor cell growths in culture are dis-
cussed. Of these derivatives, 2-phenyl-6-pyrrolidinyl-4-quinoline
sulfonate analogues 10 f, 10g, and 10k, and 4’-nitrophenyl sul-
fonate 10m exhibit superior cytotoxicity over other sulfonates.
The antiproliferative activities of these compounds correlate
well with their abilities to induce mitotic arrest and apoptosis.
Mechanistic studies indicate that they target the vinblastine
binding site of tubulin and inhibit cellular tubulin polymeri-
zation. Hence, these compounds induce the formation of aber-
rant mitotic spindles and mitotic arrest, resulting in intensive
apoptosis. The tested compounds were shown to be poor sub-
strates for membrane multidrug resistance transporters. The
present studies suggest that these newly synthesized com-
pounds are promising tubulin polymerization inhibitors and
are worthy of further investigation as antitumor agents.
Introduction
Microtubules are long, rigid, cylindrical tubes with walls that
are formed by the assembly of a- and b-tubulin subunits. Most
microtubules undergo continuous polymerization and depoly-
merization through the addition of tubulin subunits at one
end and dissociation at the other end. Microtubules are essen-
tial for maintaining cell shape and polarity, the intracellular
transport of vesicles and organelles, and the beating of cilia
and flagella.[1,2] During eukaryotic cell division, microtubules
form mitotic spindles, which align replicated chromosomes to
the equatorial plane and mediate the subsequent segregation
of chromosomes to the two daughter cells.[3] As microtubules
play an important role in cell division, they are potential tar-
gets for the development of chemotherapeutic drugs which
target rapidly dividing cancer cells.[4,5] These drugs interfere
with the polymerization–depolymerization properties of micro-
tubules to prevent cell cycle progression, inducing the cells to
undergo programmed cell death.[2,6,7]
and tubulin assembly by destabilizing microtubules.[2,12] In con-
trast, compounds which bind to the taxane binding site pre-
vent microtubule disassembly by stabilizing microtubules.[12]
However, owing to high toxicity, poor bioavailability, and syn-
thetic difficulty, taxanes and vinblastine have limited clinical
applications. Moreover, they induce overexpression of trans-
membrane efflux proteins that pump out antimitotic inhibitors,
resulting in decreased potency and rapid development of mul-
tidrug resistance (MDR).[13] Consequently, searching for com-
pounds which target tubulin without producing cross-resist-
ance to taxane or vinblastine is an urgent need in the develop-
ment of new anticancer agents.
Combretastatin A-4 (CA-4, 1, Figure 1), isolated from Combre-
tum caffrum,[14] exhibits potent cytotoxicity with a broad spec-
[a] Dr. R. Kakadiya, Prof. T.-L. Su
Institute of Biomedical Sciences
Academia Sinica, Taipei 11529 (Taiwan)
Fax: (+886)2-27829142
Numerous small molecules with diverse structures and
potent antitumor activity were identified as effective microtu-
bule-targeted drugs in recent years. Most of these agents are
naturally occurring products, such as vinca alkaloids (vincris-
tine, vinblastine, and vinorelbine),[8] taxanes (paclitaxel and do-
cetaxel),[9] and epothilones,[10] which have all been proven to
successfully treat a wide variety of human cancers. These
agents act by either inhibiting the polymerization of tubulin
(vinca alkaloids and colchicines) or preventing the disassembly
of microtubules (taxanes and epothilones). Accordingly, three
important binding domains on b-tubulin, namely the vinca
binding site, the taxane binding site, and the colchicine bind-
ing site, have been identified.[11] Compounds which bind to the
vinca or colchicine binding sites inhibit cancer cell proliferation
E-mail: tlsu@ibms.sinica.edu.tw
[b] Y.-C. Wu, H.-H. Kuo, Dr. L.-H. Yih
Institute of Cellular and Organismic Biology
Academia Sinica, Taipei 11529 (Taiwan)
Fax: (+886)2-27858059
E-mail: lhyih@gate.sinica.edu.tw
[c] Dr. H. Dong, Dr. T.-C. Chou
Preclinical Pharmacology Core Laboratory
Molecular Pharmacology and Chemistry Program
Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (USA)
[d] Prof. T.-L. Su
Graduate Institute of Pharmaceutical Chemistry
China Medical University, Taichung (Taiwan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100121.
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Among antimitotic CA-4 ana-
logues, Gwaltney et al.[26] syn-
thesized a series of CA-4-like
sulfonates by introducing a sul-
fonate functional group as a
tether between the two aryl
rings (3–5, Figure 1), which are
structural features of CA-4.
Many of the sulfonates were re-
ported to have excellent antitu-
mor activities as well as inhibi-
tory effects against tubulin
polymerization. Structure–activi-
ty relationship (SAR) studies of
CA-4-like sulfonates demon-
strated that replacement of the
cis olefin and the aryl moieties
with a sulfonate functionality
and heterocyclic ring, respec-
tively, are well tolerated. The
orientation of the sulfonate
group relative to the two aryl
rings makes little difference in
antitumor activity. For example,
compound 3 (Figure 1) has a
sulfonate moiety adjacent to
the 3,4,5-trimethoxyphenyl ring
and exhibits greater inhibitory
activity than corresponding
compound
4 toward human
colon adenocarcinoma HCT-15
cell growth in vitro, with IC50
values of 36 and 250 nm, re-
spectively. However, compound
5, which has a 3’-amino-4’-me-
thoxy-substituted pattern was
shown to be the most potent
cell growth inhibitor, with IC50
values of 4.1 and 2.7 nm against
HCT-15 and H460 cells, respec-
Figure 1. Structure of combretastatin A-4 (CA-4, 1), water-soluble prodrug CA-4P (2), sulfonate analogues 3–7, 2-
phenylquinolones 8 and 9, and the recently generated 2-substituted quinoline sulfonates 10a–n.
trum of antitumor activity.[15–17] CA-4 and related derivatives are
microtubule inhibitors which target the colchicine binding site
and exhibit low toxicity profiles.[18,19] Despite its potent cyto-
toxic and antitubulin activities, CA-4 failed to exhibit potent
antitumor efficacy in animal models due to its limited water
solubility (poor bioavailability).[20] Accordingly, the water-solu-
ble prodrug, CA-4 disodium phosphate (2, CA-4-DP, Figure 1),
was synthesized and found to have potent antiangiogenic and
tubulin inhibition effects.[21,22] This compound is currently un-
dergoing phase I/II clinical trials.[23,24] Because of its interesting
antitumor activity, a number of CA-4 analogues have been syn-
thesized with modifications to ring A, ring B, and the bridge.[25]
It was also demonstrated that the cis isomer is more cytotoxic
than the corresponding trans isomer. Although none of the
compounds with a modified bridge exhibit more potent bioac-
tivity than CA-4, some analogues still retain high tubulin poly-
merization activity and cytotoxicity.
tively. CA-4 exhibited IC50 values of 1.7 and 3 nm, respectively,
against the same cell lines. Recently, Zuse et al.[27] synthesized
sulfonate derivatives of anthracenone (6, Figure 1) and naph-
thothiophenone (7), which can be considered anthracenoid an-
alogues of CA-4. These derivatives displayed potent cytotoxici-
ty and microtubule-disrupting activity, and are most likely to
interact with tubulin at the colchicine binding site.
The 2-phenyl-4-quinolones are another group of molecules
which display anticancer activity through the inhibition of tu-
bulin polymerization. Kuo, Lee, and colleagues synthesized a
series of 2-phenyl-4-quinolone derivatives,[28–32] including NSC
664171 (8, Figure 1) and CHM-1 (9, Figure 1), which were
shown to have potent antitumor activity. These agents inhibit-
ed the growth of NCI tumor cell lines within a small range of
nanomolar concentrations. Both compounds are potent inhibi-
tors of tubulin polymerization and have cytotoxicities similar
to that of CA-4.
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As previously mentioned, the sulfonate group can
replace the cis olefin bridge of CA-4 while preserving
cytotoxicity. However, the two moieties attached to
the sulfonate functionality can be varied, so we de-
signed and synthesized a series of 2-substituted-qui-
nolin-4-yl-benzenesulfonates for antitumor evalua-
tion which, in theory, could be enzymatically hydro-
lyzed to release the biologically active quinolones.
The results show that the newly synthesized quino-
line sulfonates exhibit significant cytotoxicity against
human lymphoblastic leukemia and inhibit growth
of various solid tumor cells in culture. Additionally,
we found that these agents target the vinblastine
site of tubulin and inhibit cellular tubulin polymeri-
zation. Herein, we report the antitumor activity and
mechanism of action of these new sulfonates.
Results and Discussion
Chemistry
The general method for the synthesis of 2-substitut-
ed-quinolin-4-yl-benzenesulfonates 10a–n is outlined
in Scheme 1. 4-Methoxybenzenesulfonyl chloride
(12b), 4-nitrobenzenesulfonyl chloride (12c), 4-hy-
droxyquinoline (13a), and 2-methyl-4-hydroxyquino-
line (13b) are commercially available. 3,4,5-Trimethoxybenze-
nesulfonyl chloride (12a)[33] and 2-substituted-4-hydroxyquino-
lines (13b–m) were prepared following the previously de-
scribed methods.[28–30] The desired sulfonates (10a–n) were
prepared in good yield by combining 2-substituted-4-hydroxy-
quinolines 13a–n with the appropriate benzenesulfonyl chlor-
ides (12a–c) in the presence of potassium carbonate in anhy-
drous DMF.
Scheme 1. Reagents and conditions: a) NaNO2/HCl-AcOH, CH3CN, 08C, 30 min; b) SO2 in
AcOH/CuCl, 2 h; c) Anhyd. DMF/K2CO3, 2 h.
Biological results
In vitro cytotoxicity
The antiproliferative activities of the newly synthesized sulfo-
nate derivatives (10a–n) against human lymphoblastic leuke-
mia (CCRF-CEM) and human solid tumor cell growth in culture
are summarized in Table 1. Conclusions were made regarding
the SAR for these analogues, based on the IC50 value of each
Table 1. Cytotoxicity of 2-substituted quinoline-4-yl-benzenesulfonates (10a–n) in CCRF-CEM, the vinblastine- and taxol-resistant sub-cell lines, and various
human solid tumor cell lines.
IC50 [mm][a]
HCT-116
Compd
CCRF-CEM
ND[d]
CCRF-CEM/VBL[b]
ND
CCRF-CEM/Taxol[b]
ND
HeLa-S3
H460
HT29
PC3
10a
10b
10c
10d
10e
10 f
10g
10h
10i
10j
10k
10l
10m
10n
ND
9.32Æ0.06
8.53Æ0.05
1.88Æ0.04
3.35Æ0.07
1.05Æ0.04
0.26Æ0.03
0.31Æ0.04
0.64Æ0.05
1.21Æ0.05
1.84Æ0.04
0.37Æ0.04
0.31Æ0.03
0.33Æ0.03
ND
ND
ND
ND
ND
ND
2.98Æ0.44
2.34Æ0.002
2.39Æ0.002
2.00Æ0.01
0.28Æ0.001
0.75Æ0.07
0.38Æ0.01
0.50Æ0.02
1.80Æ0.06
0.81Æ0.01
1.46Æ0.02
0.47Æ0.001
ND
4.26Æ0.01 [1.4ꢁ][c] 5.12Æ0.001 [1.7ꢁ][c] 3.43Æ0.06
6.27Æ0.40 [2.7ꢁ] 3.55Æ0.05 [1.5ꢁ]
3.96Æ0.07 [1.7ꢁ] 3.87Æ0.04 [1.6ꢁ]
5.24Æ0.01 [2.6ꢁ] 3.28Æ0.14 [1.6ꢁ]
1.17Æ0.11 [4.2ꢁ] 1.28Æ0.01 [4.6ꢁ]
1.32Æ0.004 [1.8ꢁ] 1.13Æ0.04 [1.5ꢁ]
0.99Æ0.01 [2.5ꢁ] 0.59Æ0.02 [1.5ꢁ]
1.28Æ0.01 [2.6ꢁ] 1.04Æ0.01 [2.1ꢁ]
8.46Æ0.05 [4.7ꢁ] 3.98Æ0.11 [2.2ꢁ]
1.67Æ0.002 [2.1ꢁ] 1.05Æ0.01 [1.3ꢁ]
2.63Æ0.01 [1.8ꢁ] 2.31Æ0.02 [1.6ꢁ]
1.70Æ0.04 [3.6ꢁ] 0.91Æ0.04 [1.9ꢁ]
5.20Æ0.16
4.34Æ0.02
5.35Æ0.07
0.83Æ0.003
1.25Æ0.01
1.07Æ0.03
1.17Æ0.001
6.39Æ0.09
0.99Æ0.01
3.12Æ0.04
1.34Æ0.02
ND
6.80Æ0.06
11.54Æ0.06
3.11Æ0.04
0.34Æ0.03
0.58Æ0.04
2.47Æ0.06
ND
6.26Æ0.05
0.44Æ0.04
0.47Æ0.04
0.79Æ0.06
ND
4.41Æ0.03
7.84Æ0.15
2.60Æ0.04
0.43Æ0.09
0.60Æ0.09
3.07Æ0.02
ND
4.00Æ0.20
0.77Æ0.09
0.66Æ0.07
0.90Æ0.07
ND
5.42Æ0.03
8.75Æ0.06
3.40Æ0.03
0.51Æ0.06
0.59Æ0.06
1.50Æ0.08
ND
4.07Æ0.06
0.68Æ0.05
0.64Æ0.05
0.79Æ0.04
ND
ND
ND
>100
vinblastine 0.0007Æ0.0001 0.50Æ0.12 [680ꢁ] 0.08Æ0.01 [106ꢁ]
ND
ND
0.0006Æ0.00003 0.0014Æ0.0004 0.002Æ0.0001 0.001Æ0.0001
0.003Æ0.00001 0.004Æ0.00002 0.006Æ0.0001 0.004Æ0.0001
taxol
0.0012Æ0.0001 1.27Æ0.05 [980ꢁ] 0.43Æ0.04 [358ꢁ]
[a] Data represent the mean ÆSD of each compound from three to six independent experiments. [b] CCRF-CEM/VBL and CCRF-CEM/Taxol are sub-cell
lines of CCRF-CEM cells with 680-fold resistance to vinblastine and 358-fold resistance to taxol, respectively, relative to the IC50 value of the parent cell line.
[c] Numbers in brackets are measures of cross-resistance as determined by comparison with the corresponding IC50 value of the parent cell line. [d] Not de-
termined.
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compound in CCRF/CEM cells. Within the series of 3,4,5-trime-
thoxyphenyl sulfonate derivatives (10a–i), the 2-substututed
phenyl derivatives (10c–i, IC50 =0.28–2.39 mm) are more cyto-
toxic than the C2-unsubstituted compound (10a, IC50 =
9.32 mm) and 2-methyl-substituted compound (10b, IC50 =
2.98 mm). The 6,7-methylenedioxy-2-phenylquinoline deriva-
tives (10c–e) can be ranked in the following order with respect
to potency: 2[2’-fluorophenyl] (10e) > 2-phenyl (10c) > 2[3’-
methoxyphenyl] (10d). However, 2[3’-methoxyphenyl] deriva-
tive 10 f is more cytotoxic than 2[2’-fluorophenyl] 10g among
the 6-pyrrolidinyl quinolines. Additionally, among the series of
3’-methoxyphenyl derivatives, 6-chloroquinoline derivative 10h
exhibits greater cytotoxicity than the corresponding 6-fluoro-
quinoline derivative (10i).
most potent derivatives, with an IC50 value of 0.47 mm against
CCRF/CEM cell growth in culture. It should be noted that 2-[2’-
fluorophenyl]-6-pyrrolinylquinoline derivatives exhibit signifi-
cant cytotoxicity with both 3’,4’,5’-trimethoxyphenyl (10 f, and
10g) and 4-methoxyphenyl sulfonates (10k and 10l). We fur-
ther evaluated the cytotoxicity of the newly synthesized sulfo-
nates in human solid tumor cell lines (HCT-116, HeLa-S3, H460,
HT29, and PC3). Compounds 10 f, 10g, 10k, and 10m exhibit-
ed a broad spectrum of antitumor activity, with IC50 values
ranging from 0.28–1.34 mm against the cell lines tested. It
should be noted that compounds without a C2 substituent,
10a and 10n, exhibit less or no cytotoxicity, respectively, than
the other compounds, indicating the importance of a substitu-
ent at the C2 position.
The 4-methoxyphenyl sulfonate derivative series (10j–l) can
be ranked in order of decreasing potency as follows: 10k >
10l > 10j. The 4’-nitrophenyl sulfonate (10m) was one of the
One major limitation of anticancer drugs is acquired multi-
drug resistance (MDR). We used CCRF/CEM cells, the vinblas-
tine-resistant subline (CCRF-CEM/VBL, 680-fold resistance to
Figure 2. A) and B) Quinoline sulfonates induce mitotic arrest and apoptosis: Representative flow cytometry data for untreated and compound 10m-treated
(1 mm for 24 or 72 h) cells stained with PI for DNA labeling and with an antibody against phospho-histone H3 (p-H3) to label mitotic cells (A) or with an anti-
body against cleaved PARP to label apoptotic cells (B). Rectangles indicate p-H3-positive or cleaved PARP-positive cells; the percentage of mitotic or apoptotic
cells is indicated in bold. C) and D) Quinoline sulfonates induce accumulation of mitotic cells: HeLa-S3 cells were treated with 0.5–2 mm quinoline sulfonates
or 0.5–2 nm taxol or vinblastine (Vin) for 24 (C) and 72 h (D), then cell cycle distribution (C) and apoptosis (D) were analyzed as described in the Experimental
Section. Data are the mean ÆSD of four independent experiments.
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Tubulin Polymerization Inhibitors
vinblastine in vitro), and the
taxol-resistant subline (CCRF-
CEM/taxol, 358-fold resistance to
taxol in vitro) to evaluate wheth-
er the compounds could over-
come the MDR developed in
these sublines (Table 1). The re-
sulting IC50 values showed that
the sulfonates are 1.4- to 4.7-
fold less cytotoxic in the resist-
ant sublines than in parent
CCRF/CEM cells. This indicates
that the tested sulfonates have
little or no cross-resistance to
vinblastine or taxol and are not
good substrates for membrane
MDR transporters.
Induction of mitotic arrest and
apoptosis
Six of the compounds exhibiting
the highest levels of cytotoxicity
in regard to inhibition of cancer
cell growth (10 f, 10g, 10h,
10k, 10l, and 10m) were exam-
ined for their effects on cell
cycle progression and apoptosis
Figure 3. Quinoline sulfonates induce abnormal mitotic spindles: Representative immunofluorescence images
showing abnormal spindles in mitotic cells arrested by quinoline sulfonates, taxol, or vinblastine. HeLa-S3 cells
seeded on glass cover slips were treated for 14 h with A) vehicle, B) 10 nm vinblastine, C) 10 nm taxol, or D) 1 mm
quinoline sulfonates, and stained with an anti-a-tubulin antibody to detect mitotic spindles (left), with an anti-g-
tubulin antibody to detect centrosomes (center), and with DAPI to detect chromosomes (right).
induction in HeLa-S3 cells by flow cytometry analysis of phos-
pho-histone H3 (a mitosis marker) and cleaved poly(ADP-
ribose) polymerase (PARP; an apoptosis marker). Results
showed that drug treatment induced a significant increase in
mitotic cells at 24 h (Figure 2A and C) and apoptosis at 72 h
(Figure 2B and D), indicating that cells were first arrested at
mitosis and then underwent apoptosis following treatment
with sulfonate derivatives. Of the compounds tested, 10h,
which exhibits relatively low cytotoxicity (IC50 =0.64 mm in
HeLa-S3 cells), induced low levels of mitotic arrest and apopto-
sis at 0.5 and 1 mm, respectively. The other compounds (10 f,
10g, 10k, 10l, and 10m) have higher cytotoxicity (IC50: 0.26–
0.37 mm in HeLa-S3 cells) and induced higher levels of mitotic
arrest and apoptosis at the same concentrations. These results
indicate that the ability of these drugs to induce mitotic arrest
and apoptosis correlate well with their antiproliferative activi-
ties.
Antimitotic effects of sulfonates
As selected sulfonates (1–2 mm concentration) induced nearly
80% of cells arrested at mitosis 24 h after treatment (Fig-
ure 2C), their effects on mitotic spindles were evaluated. The
results showed that the bipolar spindles are organized in an
oval shape, while the chromosomes are aligned at metaphase
plates in all metaphases from untreated HeLa-S3 cells (Fig-
ure 3A). In contrast, all of the mitotic cells arrested using 1 mm
of the tested compounds (10 f, 10g, 10h, 10k, 10l, and 10m)
contained highly abnormal mitotic spindles with multiple
Figure 4. Quinoline sulfonates inhibit cellular tubulin polymerization: HeLa-
S3 cells were treated with 1 mm sulfonates, 10 nm vinblastine, or 10 nm taxol
for 24 h. Mitotic cells were removed by shaking, and equal amounts of cells
were lysed and analyzed as described in the Experimental Section. S, soluble
a-tubulin; P, polymerized a-tubulin. Ratios to the right of each blot indicate
percentage of polymerized or soluble tubulin as determined by dividing the
densitometric value for each tubulin fraction by the total tubulin content
(polymerized + soluble tubulin). Normal mitotic cells were obtained from
cultures released after 10 h from a double-thymidine block.
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L.-H. Yih, T.-L. Su, et al.
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poles, shortened and dispersed microtubule fibers, and mis-
segregated chromosomes (Figure 3D). This pattern is similar to
that shown for vinblastine-arrested mitotic cells (Figure 3B),
and indicates that the sulfonate derivatives disrupt the assem-
bly of bipolar spindles and alter chromosome segregation, in-
ducing mitotic arrest and apoptosis.
which exhibits relatively low cytotoxicity in HeLa-S3 cells, has
no effect on the intrinsic tryptophan fluorescence of tubulin
(Figure 5A) and does not compete for the vinblastine binding
site of tubulin as the other sulfonate derivatives do (Figure 5C).
These findings provide evidence that the newly synthesized
sulfonate derivatives interact directly with tubulin through the
vinblastine site but not the colchicine site. Additionally, the re-
sults suggest that the affinity of drug–tubulin binding may not
have correlate directly with drug cytotoxicity.
To ascertain the mechanism of action for the sulfonates, we
evaluated their effects on cellular microtubules using the cellu-
lar microtubule stabilization assay. The results showed that tu-
bulin extracted from synchronized mitotic cells is primarily in
the polymerized form (Figure 4). In contrast, up to 80% of tu-
bulin extracted from sulfonate-arrested mitotic cells is in the
soluble form, as observed in vinblastine-arrested mitotic cells.
Tubulin extracted from taxol-arrested mitotic cells is primarily
polymerized. Therefore, taxol stabilizes and vinblastine desta-
bilizes tubulin polymerization, as expected, while the sulfo-
nates inhibit cellular tubulin polymerization.
Conclusions
We have synthesized a series of 2-substituted quinolin-4-yl-
benzenesulfonates for evaluation of in vitro cytotoxicity. Of
these derivatives, compounds with a 6-pyrrolidinyl functionali-
ty (e.g., 10 f, 10g, and 10k) and 4’-nitrophenyl sulfonate
(10m) exhibit significant antiproliferative activities against all
tumor cell lines tested. We found that these sulfonates are
able to induce a significant increase in mitotic arrest and apop-
tosis. Studies regarding the effects of these compounds on mi-
totic spindles revealed that the synthesized sulfonates interfere
with assembly and function of mitotic spindles and block mito-
sis progression, thereby inducing apoptosis. Previous studies
showed that sulfonate analogues of CA-4 bind to the colchi-
cine binding domain of tubulin.[26] In contrast, the compounds
synthesized in this study bind to the vinblastine, but not the
colchicines, domain. Among known tubulin polymerization in-
hibitors, vinblastine and colchicine binding sites, as well as
their mechanisms of action of these compound, have been
studied thoroughly. As mentioned previously, colchicine deriva-
tives have limited applications in cancer chemotherapy due to
their toxicity, which results in hemorrhaging and necrosis.[37]
Most drugs that interact with tubulin are isolated from natural
products (e.g., vinca alkaloids and related analogues); only a
few inhibitors are derived from synthetic compounds.[38,39] Al-
though vinca alkaloids and taxol analogues are important cate-
gories of antimitotic drugs, both rapidly develop MDR due to
overexpression of P-glycoprotein or mutated tubulin, which
decreases their therapeutic efficacy. The newly synthesized sul-
fonates, however, have no cross-resistance to the MDR cell
lines evaluated, suggesting that it may be possible to optimize
the antitumor efficacy of these sulfonates via structural modifi-
cation to generate more promising antitubulin agents for clini-
cal application.
Interaction of sulfonates with tubulin and targeting of the
vinblastine binding site
A majority of compounds that inhibit tubulin polymerization
bind to tubulin at either the colchicine site or the vinblastine
site.[6] Therefore, through competitive binding assays, we ex-
amined whether or not sulfonate derivatives interact with tu-
bulin directly by binding to these sites. The binding of sulfo-
nates to tubulin was first investigated by measuring the effects
of these drugs on the intrinsic tryptophan fluorescence of tu-
bulin. Compound 10h was not included because it has strong
auto fluorescence, which interferes with measurement. As
shown in Figure 5A, all of the sulfonate derivatives evaluated
(10 f, 10g, 10k, 10l, and 10m) decrease the intrinsic trypto-
phan fluorescence of tubulin similarly to vinblastine, indicating
that these drugs interact with tubulin. The intrinsic fluores-
cence of colchicine increases upon binding to tubulin,[34] which
was used as an indicator for the competition of sulfonate de-
rivatives with colchicine in tubulin binding. Our results showed
that pre-incubation of tubulin with each sulfonate derivative
does not decrease the fluorescence of the colchicine–tubulin
complex (Figure 5B), indicating that these agents have no
effect on the colchicine–tubulin interaction. In contrast, pre-in-
cubation of tubulin with colcemid, a colchicine analogue with
very low fluorescence upon binding to tubulin,[35] significantly
decreased fluorescence of the colchicine–tubulin complex.
These results indicate that the tubulin binding site of these sul-
fonate derivatives is different from that of colchicine.
Experimental Section
The fluorescence of BODIPY FL-vinblastine, a fluorescent de-
rivative of vinblastine, increases significantly upon binding to
tubulin.[36] Therefore, BODIPY FL-vinblastine was employed to
detect whether the tested sulfonate derivatives could bind to
the vinblastine binding site. Pre-incubation of tubulin with
each of the sulfonate derivatives or vinblastine significantly de-
creases the fluorescence of BODIPY FL-vinblastine (Figure 5C),
indicating that these agents prevent the binding of BODIPY FL-
vinblastine to tubulin. Taxol, by comparison, which does not
bind to the vinblastine binding site of tubulin, has no effect on
the fluorescence of BODIPY FL-vinblastine. Compound 10a,
Chemistry: general methods
All commercial chemicals and solvents were reagent grade and
were used without further purification unless otherwise specified.
Melting points were determined using a Fargo melting point appa-
ratus and are uncorrected. Column chromatography was carried
out on silica gel G60 (70–230 mesh, ASTM, Merck; and 230–400
mesh, Silicycle Inc.). All high-resolution mass spectra were recorded
using a Bruker BioTOF II mass spectrometer using ESI in positive
ion mode. Thin-layer chromatography was performed on silica gel
G60 F254 (Merck) with short-wavelength UV light for visualization.
1124
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ChemMedChem 2011, 6, 1119 – 1129

Tubulin Polymerization Inhibitors
Figure 5. Characterization of binding of quinoline sulfonates to tubulin: A) Quinoline sulfonates decrease the intrinsic tryptophan fluorescence of tubulin. The
excitation wavelength was 295 nm, and one of four experiments is shown. B) Quinoline sulfonates do not alter fluorescence of the tubulin–colchicine com-
plex. Tubulin (5 mm) was first incubated with 20 mm each quinoline sulfonate, 20 mm colcemid, or 20 mm vinblastine at 378C for 45 min, then 20 mm colchicine
was added to each of the mixtures. Fluorescence was measured after incubation for 20 min at 378C. Excitation and emission wavelengths were 350 and
435 nm, respectively. Data are the average of two independent experiments. C) Quinoline sulfonates inhibit binding of vinblastine to tubulin. Tubulin (3 mm)
was first incubated with 20 mm quinoline sulfonates, 20 mm taxol, or 20 mm vinblastine at 378C for 45 min, then 3 mm BODIPY FL-vinblastine was added to
each of the mixtures. Fluorescence was measured after incubation for 20 min at 378C. The excitation wavelength was 490 nm, and one of four experiments is
shown.
All reported yields correspond to isolated yields after chromatogra-
phy or crystallization. Elemental analyses were done using a Her-
sample volume: 10 mL; column T=278C; l=254 nm. The purity of
all compounds was ꢀ95% based on two analytical HPLC methods.
1
aeus CHN-O Rapid instrument. H NMR and 13C NMR spectra were
recorded on a 600 MHz, Brucker AVANCE 600 DRX and 400 MHz,
Brucker Top-Spin spectrometers in the solvents indicated. Chemical
shifts (d) are reported in ppm relative to (CH3)4Si. High-perfor-
mance liquid chromatography (HPLC) purity analyses of synthe-
sized compounds were recorded on a Hitachi D-2000 Elite instru-
ment with a Mightysil RP-18 GP 250–4.6 column (5 mm); mobile
phase: 90% CH3CN, 5% THF, and 5% H2O over 25 min or 90%
CH3CN and 10% MeOH over 25 min; flow rate: 1 mLminÀ1; injected
Starting materials for preparing 2-substituted-quinolin-4-yl-ben-
zenesulfonates: 4-methoxybenzenesulfonyl chloride (12b), 4-nitro-
sulfonyl chloride (12c), quinolin-4(1H)-one (13a), and 2-methylqui-
nolin-4(1H)-one (13b) are commercially available; 3,4,5-trimethoxy-
phenylsulfonyl chloride (12a) was prepared as previously de-
scribed.[33] 2-Substituted 4-hydroxyquinolines were prepared fol-
lowing methods described in literature.[28–30]
ChemMedChem 2011, 6, 1119 – 1129
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www.chemmedchem.org
1125

L.-H. Yih, T.-L. Su, et al.
MED
Quinolin-4-yl-3,4,5-trimethoxybenzenesulfonate (10a): Potassium
carbonate (0.55 g, 4 mmol) was added to a solution of quinolin-
4(1H)-one 13a (0.29 g, 2 mmol) in anhydrous DMF (10 mL). The
mixture was sonicated for 20 min, then cooled to 08C. A solution
of 3,4,5-trimethoxybenzenesulfonyl chloride (12a, 0.69 g, 4 mmol)
in anhydrous DMF (5 mL) was added dropwise. The resulting solu-
tion was stirred at room temperature for 2 h. The solvent was
evaporated to dryness in vacuo, and the residue was dissolved in
CH2Cl2 and washed with H2O. The organic phases were combined,
dried over anhydrous Na2SO4, filtered, and evaporated in vacuo.
The resulting crude solid was recrystallized from EtOH to give 10a.
6-(2-Fluorophenyl)-[1,3]dioxolo[4,5-g]quinolin-8-yl-3,4,5-trime-
thoxybenzenesulfonate (10e): Compound 10e was prepared
from 6-(2-fluorophenyl)-[1,3]dioxolo[4,5-g]quinolin-8(5H)-one (13e,
1.13 g, 4 mmol) and 12a (1.60 g, 6 mmol). Yield: 1.87 g, 89%; mp:
1
176–1778C; H NMR ([D6]DMSO): d=3.72 (s, 3H, OMe), 3.77 (s, 6H,
2ꢁOMe), 6.26 (s, 2H, CH2), 7.20 (s, 2H, 2ꢁArH), 7.22 (s, 1H, ArH),
7.32–7.39 (2H, m 2ꢁArH), 7.46 (s, 1H, ArH), 7.47 (s, 1H, ArH), 7.52–
7.55 (m, 1H, ArH), 8.01–8.05 ppm (m, 1H, ArH); 13C NMR
([D6]DMSO): d=56.43, 60.32, 96.44, 102.72, 105.27, 106.09, 112.35,
116.49, 117.66, 124.90, 125.95, 128.13, 130.85, 131.62, 143.08,
147.96, 148.87, 150.95, 151.84, 153.21, 158.71, 161.19 ppm; HRMS
[M+H]+ calcd for C25H20FNO8S: 514.0972, found: 514.3616; MS (EI)
m/z [M+] (rel. intensity): 513.0 (18%); Anal. calcd for C25H20FNO8S:
C 58.48, H 3.93, N 2.73, S 6.24, found: C 58.28, H 4.03, N 3.04, S
6.56.
1
Yield, 0.67 g (89%); mp: 93–948C; H NMR ([D6]DMSO): d=3.73 (s,
3H, OMe), 3.77 (s, 6H, 2ꢁOMe), 7.16 (s, 2H, 2ꢁArH), 7.45 (d, 1H,
J=4.5 Hz, ArH), 7.62 (t, 1H, J=7.6 Hz, ArH), 7.82 (t, 1H, J=7.6 Hz,
ArH), 7.90 ppm (d, 1H, J=8.4 Hz, ArH); 13C NMR (400 MHz,
[D6]DMSO): d=56.46, 60.37, 105.88, 113.15, 121.30, 121.66, 127.55,
128.23, 128.93, 130.62, 143.00, 148.47, 151.39, 152.22, 153.18 ppm;
HRMS calcd for C18H17NO6S: 376.0856 [M+H]+, found: 376.2784;
MS (EI) m/z (rel. intensity): 375.0 (29%) [M]+.
2-(3-Methoxyphenyl)-6-(pyrrolidin-1-yl)quinolin-4-yl-3,4,5-trime-
thoxybenzenesulfonate (10 f): Compound 10 f was prepared from
2-(3-methoxyphenyl)-6-(pyrrolidin-1-yl)quinolin-4(1H)-one
(13 f,
0.80 g, 2.5 mmol) and 12a (0.94 g, 3.5 mmol). Yield: 1.18 g, 85%;
mp: 168–1698C; H NMR ([D6]DMSO): d=1.98 (m, 4H, 2ꢁCH2), 3.24
The following compounds were prepared using the same synthetic
method as for 10a:
1
(m, 4H, 2ꢁCH2), 3.69 s, 9H, 3ꢁOMe), 3.86 (s, 3H, OMe), 6.33–6.32
(m, 1H, ArH), 7.04–7.02 (m, 1H, ArH), 7.10 (s, 2H, 2ꢁArH), 7.29–
7.26 (m, 1H, ArH), 7.44–7.40 (m, 1H, ArH), 7.66–7.64 (m, 2H, 2ꢁ
ArH), 7.71 (s, 1H, ArH), 7.91–7.89 ppm (m, 1H, ArH); 13C NMR
([D6]DMSO): d=24.93, 47.29, 55.17, 56.29, 60.22, 95.76, 105.86,
111.54, 114.80, 118.72, 120.40, 122.84, 128.89, 129.86, 130.11,
130.85, 139.68, 142.93, 145.88, 150.56, 151.13, 153.18, 159.74,
160.89 ppm; HRMS [M+H]+ calcd for C29H30N2O7S: 551.1851,
found: 551.4715; MS (EI) m/z [M]+ (rel. intensity): 550.2 (14%);
Anal. calcd for C29H30N2O7S: C 63.26, H 5.49, N 5.09, S 5.82, found:
C 63.25, H 5.46, N 5.26, S 6.13.
2-Methylquinolin-4-yl-3,4,5-trimethoxybenzenesulfonate (10b):
Compound 10b was prepared from 2-methylquinolin-4(1H)-one
(13b, 0.41 g, 2.56 mmol) and 12a (0.80 g, 3 mmol). Yield: 0.80 g,
80%; mp: 99–1018C; 1H NMR ([D6]DMSO): d=2.67 (s, 3H, Me),
3.67–3.84 (m, 9H, 3ꢁOMe), 7.11–7.19 (m, 2H, ArH), 7.33 (s, 1H,
ArH), 7.47–7.52 (m, 1H, ArH), 7.68–7.86 (m, 2H, ArH), 7.89–
8.04 ppm (m, 1H, ArH); 13C NMR ([D6]DMSO): d=24.65, 56.49,
60.35, 102.94, 105.97, 113.71, 120.06, 121.20, 123.56, 126.72, 128.19,
130.83, 143.08, 151.96, 153.16, 159.97 ppm; HRMS [M+H]+ calcd
for C19H19NO6S: 390.1011, found: 390.2993; MS (EI) m/z [M]+ (rel. in-
tensity): 388.9 (17%); Anal. calcd for C19H19NO6S: C 58.60, H 4.92, N
3.60, S 8.23, found: C 58.57, H 4.70, N 3.62, S 8.57.
2-(2-Fluorophenyl)-6-(pyrrolidin-1-yl)quinolin-4-yl-3,4,5-trime-
thoxybenzenesulfonate (10g): Compound 10g was prepared
from 2-(2-fluorophenyl)-6-(pyrrolidin-1-yl)quinolin-4(1H)-one (13g,
0.90 g, 3.0 mmol) and 12a (1.20 g, 4.5 mmol). Yield: 1.23 g, 76%;
mp: 174–1758C; 1H NMR ([D6]DMSO): d=1.99, (m, 4H, 2ꢁCH2),
3.28, (m, 4H, 2ꢁCH2), 3.69, (s, 3H, OMe), 3.71, (s, 6H, 2ꢁOMe),
6.38–6.39, (m, 1H, ArH), 7.13, (s, 2H, 2ꢁArH), 7.30–7.38, (m, 3H, 3ꢁ
ArH), 7.48–7.53, (m, 1H, ArH), 7.56, (s, 1H, ArH), 7.90–7.92, (m, 1H,
ArH), 8.04–8.08 ppm (m, 1H, ArH); 13C NMR ([D6]DMSO): d=24.90,
47.30, 56.24, 60.19, 95.48, 105.79, 114.23, 116.18, 120.52, 122.71,
124.78, 126.44, 128.75, 130.12, 130.60, 130.93, 142.96, 146.10,
146.94, 150.34, 153.16, 158.64, 161.11 ppm; HRMS [M+H]+ calcd
for C28H27FN2O6S: 539.1652, found: 539.4455; MS (EI) m/z [M]+ (rel.
intensity): 538.1 (8%); Anal. calcd for C28H27FN2O6S: C 62.44, H 5.05,
N 5.20, S 5.95, found: C 62.16, H 4.90, N 5.32, S 5.92.
6-Phenyl-[1,3]dioxolo[4,5-g]quinolin-8-yl-3,4,5-trimethoxybenze-
nesulfonate (10c): Compound 10c was prepared from 6-phenyl-
[1,3]dioxolo[4,5-g]quinolin-8(5H)-one (13c, 0.80 g, 3 mmol) and
12a (1.20 g, 4.5 mmol). Yield: 1.09 g, 74%; mp: 142–1438C;
1H NMR ([D6]DMSO): d=3.74 (s, 3H, OMe), 3.75 (s, 6H, 2ꢁOMe),
6.23 (s, 2H, CH2), 7.12 (s, 1H, ArH), 7.19 (s, 2H, 2ꢁArH), 7.45 (s, 1H,
ArH), 7.55–7.49 (m, 3H, 3ꢁArH), 7.64 (s, 1H, ArH), 8.08–8.06 ppm
(m, 1H, ArH); 13C NMR ([D6]DMSO): d=56.46, 60.33, 96.48, 102.55,
105.24, 106.22, 109.36, 117.60, 126.68, 127.14, 128.18, 128.67,
128.80, 129.64, 137.73, 143.14, 147.93, 148.41, 151.58, 152.49,
153.18, 154.69 ppm; HRMS [M+H]+ calcd for C25H21NO8S: 496.1066,
found: 496.3615; MS (EI) m/z [M]+ (rel. intensity): 495.0 (39%);
Anal. calcd for C25H21NO8S: C 60.60, H 4.27, N 2.83, S 6.47, found: C
60.52, H 4.04, N 3.12, S 6.52.
6-(3-Methoxyphenyl)-[1,3]dioxolo[4,5-g]quinolin-8-yl-3,4,5-trime-
thoxybenzenesulfonate (10d): Compound 10d was prepared
6-Chloro-2-(3-methoxyphenyl)quinolin-4-yl-3,4,5-trimethoxyben-
zenesulfonate (10h): Compound 10h was prepared from 6-
from
6-(3-methoxyphenyl)-[1,3]dioxolo[4,5-g]quinolin-8(5H)-one
chloro-2-(3-methoxyphenyl)quinolin-4(1H)-one
(13h,
1.01 g,
(13d, 1.47 g, 5 mmol) and 12a (2.00 g, 7.5 mmol). Yield: 2.10 g,
81%; mp: 151–1528C; 1H NMR ([D6]DMSO): d=3.74 (s, 3H, OMe),
3.76 (s, 6H, 2ꢁOMe), 3.85 (s, 3H, OMe), 6.20 (s, 2H, CH2), 7.06–7.08
(m, 1H, ArH), 7.13 (s, 1H, ArH), 7.18 (s, 2H, 2ꢁArH), 7.42–7.45 (m,
2H, 2ꢁArH), 7.62–7.63 ppm (m, 3H, 3ꢁArH); 13C NMR ([D6]DMSO):
d=55.19, 56.47,60.33, 96.52, 102.58, 105.29, 106.22, 109.65, 111.98,
115.35, 117.77, 119.05, 128.18, 129.93, 139.20, 143.13, 147.86,
148.49, 151.62, 152.48, 153.19, 154.47, 159.73 ppm; HRMS [M+H]+
calcd for C26H23NO9S: 526.1172, found: 526.3872; MS (EI) m/z [M]+
(rel. intensity): 525.1 (17%); Anal. calcd for C26H23NO9S: C 59.42, H
4.41, N 2.67, S 6.10, found: C 59.64, H 4.36, N 2.98, S 6.25.
3.54 mmol) and 12a (1.41 g, 5.3 mmol). Yield: 1.20 g, 72%; mp:
132–1338C; H NMR ([D6]DMSO): d=3.72 (s, 9H, 3ꢁOMe), 3.87 (s,
1
3H, OMe), 7.13 (m, 3H, ArH), 7.51–7.47 (m, 1H, ArH), 7.72–7.75 (m,
3H, ArH), 7.82 (m, 1H, ArH), 7.89 (s, 1H, ArH), 8.13–8.11 ppm (m,
1H, ArH); 13C NMR ([D6]DMSO): d=55.27, 56.46, 60.24, 106.23,
112.38, 112.68, 116.18, 119.58, 120.16, 121.68, 127.78, 130.10,
131.26, 131.50, 131.96, 138.71, 143.30, 147.45, 152.40, 153.22,
157.40, 159.80 ppm; HRMS [M+H]+ calcd for C25H22ClNO7S:
516.0884, found: 516.3555; MS (EI) m/z [M+] (rel. intensity): 515.0
(7%); Anal. calcd for C25H22ClNO7S: C 58.20, H 4.30, N 2.71, S 6.21,
found: C 58.20, H 4.44, N 2.80, S 6.46.
1126
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ChemMedChem 2011, 6, 1119 – 1129

Tubulin Polymerization Inhibitors
6-Fluoro-2-(3-methoxyphenyl)quinolin-4-yl-3,4,5-trimethoxyben-
zenesulfonate (10i): Compound 10i was prepared from 6-fluoro-2-
(3-methoxyphenyl)quinolin-4(1H)-one (13i, 0.50 g, 1.75 mmol) and
12a (0.71 g, 2.65 mmol). Yield: 0.72 g, 78%; mp: 126–1288C;
1H NMR ([D6]DMSO): d=3.68–3.77 (m, 9H, 3ꢁOMe), 3.86 (s, 3H,
OMe), 7.07–7.22 (m, 3H, ArH), 7.43–7.56 (m, 2H, ArH), 7.65–7.80 (m,
3H, ArH), 7.92 (s, 1H, ArH), 8.14–8.24 ppm (m, 1H, ArH); 13C NMR
([D6]DMSO): d=55.68, 56.15, 60.29, 103.33, 105.19, 106.10, 112.29,
112.54, 114.54, 119.92, 121.61, 122.18, 128.29, 130.43, 131.52,
141.16, 143.29, 144.80, 151.59, 153.18, 159.92, 160.82 ppm; HRMS
[M+H]+ calcd for C25H22FNO7S: 500.1180, found: 500.3733; MS (EI)
m/z [M]+ (rel. intensity): 498.9 (28%); Anal. calcd for C25H22FNO7S:
C 60.11, H 4.44, N 2.80, S 6.42, found: C 60.20, H 4.40, N 2.71, S
6.57.
1.10 g, 5 mmol) and 12c (1.86 g, 7.0 mmol). Yield: 1.50 g, 82%; mp:
176–1778C; 1H NMR ([D6]DMSO): d=7.56–7.64 (m, 4H, 4ꢁArH),
7.84–7.86 (m, 2H, 2ꢁArH), 7.95 (s, 1H, ArH), 8.13–8.15 (m, 1H,
ArH), 8.18–8.20 (m, 2H, 2ꢁArH), 8.35–8.37 (m, 2H, 2ꢁArH), 8.44–
8.46 ppm (m, 2H, 2ꢁArH); 13C NMR ([D6]DMSO): d=110.81, 120.44,
121.02, 123.31, 125.15, 126.90, 127.27, 127.74, 128.22, 128.98,
129.23, 130.31, 131.30, 137.46, 139.15, 149.27, 151.32, 152.88,
157.14 ppm; HRMS [M+H]+ calcd for C21H14N2O5S: 407.0702,
found: 407.2792; MS (EI) m/z [M]+ (rel. intensity): 405.9 (38%);
Anal. calcd for C21H14N2O5S: C 62.06, H 3.47, N 6.89, S 7.89, found:
C 61.91, H 3.49, N 6.92, S 7.75.
Quinolin-4-yl-4-nitrobenzenesulfonate (10n): Compound 10n
was prepared from quinolin-4(1H)-one (13a, 0.15 g, 1 mmol) and
1
12c (0.33 g, 2 mmol). Yield: 0.29 g, 88%; mp: 233–2348C; H NMR
([D6]DMSO): d=7.39 (d, 1H, J=4.92 Hz, ArH), 7.69–7.65 (m, 1H,
ArH), 7.88–7.84 (m, 1H, ArH), 7.91 (d, 1H, J=8.3 Hz, ArH), 8.10 (d,
1H, J=8.3 Hz, ArH), 8.33 (d, 2H, J=8.84 Hz, 2ꢁArH), 8.45 (d, 2H,
J=8.84 Hz, 2ꢁArH), 8.94 (d, 1H, J=4.92 Hz, ArH); 13C NMR
(400 MHz, [D6]DMSO): d=112.89, 121.05, 121.30, 125.21, 128.0,
129.10, 130.18, 130.89, 139.15, 149.59, 151.33, 151.39, 151.78 ppm;
HRMS [M+H]+ calcd for C15H10N2O5S: 331.0389, found: 331.2077;
MS (EI) m/z [M]+ (rel. intensity): 330.0 (27%). [M]+.
6-(3-Methoxyphenyl)-[1,3]dioxolo[4,5-g]quinolin-8-yl-4-methoxy-
benzenesulfonate (10j): Compound 10j was prepared from 6-(3-
methoxyphenyl)-[1,3]dioxolo[4,5-g]quinolin-8(5H)-one (13d, 1.03 g,
3.5 mmol) and 12b (1.08 g, 5.25 mmol). Yield: 1.36 g, 87%; mp:
1
171–1728C; H NMR ([D6]DMSO): d=3.85 (s, 3H, OMe), 3.57 (s, 3H,
OMe), 6.25 (s, 2H, CH2), 7.06–7.07 (m, 1H, ArH), 7.11 (s, 1H, ArH),
7.19 (d, 2H, J=8.68 Hz, 2ꢁArH), 7.46–7.42 (m, 2H, 2ꢁArH), 7.49 (s,
1H, ArH), 7.53 (s, 1H, ArH), 7.58–7.60 (m, 1H, ArH), 7.96 ppm (d,
2H, J=8.68 Hz, 2ꢁArH); 13C NMR ([D6]DMSO): d=55.22, 56.04,
96.45, 102.64, 105.38, 109.21, 112.00, 115.18, 115.34, 117.71, 119.10,
125.01, 130.00, 131.07, 139.22, 147.93, 148.60, 151.72, 152.42,
154.44, 159.71, 164.53 ppm; HRMS [M+H]+ calcd for C24H19NO7S:
466.0960, found: 466.3345; MS (EI) m/z [M]+ (rel. intensity): 465.1
(30%); Anal. calcd for C24H19NO7S: C 61.93, H 4.11, N 3.01, S 6.89,
found: C 61.64, H 4.36, N 2.88, S 6.79.
Cytotoxicity assays
In vitro cytotoxicities of the newly synthesized quinoline sulfonates
were determined in T-cell acute lymphocytic leukemia (CCRF-CEM)
and its resistant sublines (CCRF-CEM/VBL and CCRF-CEM/taxol)
using the XTT assay,[40] and human colon carcinoma (HCT-116) with
the SRB assay[41] over a 72 h incubation using a microplate spectro-
photometer as previously described.[42] After the addition of phena-
zine methosulfate–XTT solution, incubated at 378C for 6 h, absorb-
ance at 450 and 630 nm was detected using a microplate reader
(EL 340). IC50 values were determined from dose–effect relation-
ships measured with six or seven concentrations of each drug,
using the CompuSyn software by Chou and Martin[43] based on the
median-effect principle and plot.[44,45] The range of values given for
vinblastine, taxol, and cisplatin are the mean ÆSE (n=4).
2-(3-Methoxyphenyl)-6-(pyrrolidin-1-yl)quinolin-4-yl-4-methoxy-
benzenesulfonate (10k): Compound 10k was prepared from 2-(3-
methoxyphenyl)-6-(pyrrolidin-1-yl)quinolin-4(1H)-one (13 f, 0.64 g,
2 mmol) and 12b (0.62 g, 3 mmol). Yield: 0.76 g, 77%; mp: 119–
1
1208C; H NMR ([D6]DMSO): d=1.99 (m, 4H, 2ꢁCH2), 3.26 (m, 4H,
2ꢁCH2), 3.85 (s, 3H, OMe), 3.85 (s, 3H, OMe), 6.37 (s, 1H, ArH),
7.02–7.04 (m, 1H, ArH), 7.15–7.17 (m, 2H, 2ꢁArH), 7.26–7.29 (m,
1H, ArH), 7.41–7.41 (m, 1H, ArH), 7.57–7.62 (m, 3H, 3ꢁArH), 7.87–
7.93 ppm (m, 3H, 3ꢁArH); 13C NMR ([D6]DMSO): d=24.97, 47.35,
55.14, 55.95, 95.94, 110.99, 111.54, 114.73, 115.15, 118.73, 120.44,
122.70, 125.75, 129.85, 130.14, 130.72, 139.69, 142.99, 145.90,
150.49, 151.04, 159.70, 164.33 ppm; HRMS [M+H]+ calcd for
C27H26N2O5S: 491.1641, found: 491.3707; MS (EI) m/z [M]+ (rel. in-
tensity): 490.1 (12%); Anal. calcd for C27H26N2O5S: C 66.10, H 5.34,
N 5.71, S 6.54, found: C 66.18, H 5.00, N 5.73, S 6.45.
The in vitro cytotoxicity of sulfonates against human cancer cell
lines HeLa-S3 (epithelial carcinoma), H460 (large-cell lung carcino-
ma), HT29 (colon adenocarcinoma), and PC-3 (prostate adenocarci-
noma) were determined by assaying viable cell numbers using
methylthiazole tetrazolium (WST-8) (Cell Count Kit 8, Dojindo Mo-
lecular Technologies Inc., Gaithersburg, MD, USA) as previously de-
scribed.[46] Cells were seeded in a 96-well plate (3000 cells per well)
and, 24 h later, were treated with sulfonate derivatives for 72 h. At
the end of treatment, WST-8 was added to the medium and the
plates were incubated at 378C for 1 h. Cell growth was determined
by measuring the optical absorption of reduced formazan at
450 nm and expressing the result as a percentage of the absorp-
tion of the untreated control. The IC50 for each drug was deter-
mined from dose–effect relationships with six concentrations using
GraphPad PRISM ver. 5.0 (GraphPad Inc., San Diego, CA, USA).
2-(2-Fluorophenyl)-6-(pyrrolidin-1-yl)quinolin-4-yl-4-methoxy-
benzenesulfonate (10l): Compound 10l was prepared from 2-(2-
fluorophenyl)-6-(pyrrolidin-1-yl)quinolin-4(1H)-one (13g, 0.77 g,
2.5 mmol) and 12b (0.77 g, 3.75 mmol). Yield: 0.92 g, 77%; mp:
1
175–1768C; H NMR ([D6]DMSO): d=2.01 (m, 4H, 2ꢁCH2), 3.29 (m,
4H, 2ꢁCH2), 3.84 (s, 3H, OMe), 6.39–6.40 (m, 1H, ArH), 7.15–7.17
(m, 2H, ArH), 7.29–7.38 (m, 3H, 3ꢁArH), 7.48–7.53 (m, 1H, ArH),
7.56 (s, 1H, ArH), 7.88–7.93 (m, 3H, 3ꢁArH), 8.04–8.07 ppm (m, 1H,
ArH); 13C NMR ([D6]DMSO): d=25.03, 47.40, 56.00, 95.61, 113.92,
115.21, 116.29, 120.62, 122.59, 124.88, 125.66, 126.49, 130.22,
130.67, 131.01, 143.14, 146.14, 146.94, 150.32, 158.69, 161.16,
164.38 ppm; HRMS [M+H]+ calcd for C26H23FN2O4S: 479.1441,
found: 479.1382; MS (EI) m/z [M]+ (rel. intensity): 478.1 (15%);
Anal. calcd for C26H23FN2O4S: C 65.26, H 4.84, N 5.85, S 6.70, found:
C 65.29, H 4.59, N 5.85, S 6.67.
Analysis of cell cycle distribution
Cell cycle progression was monitored using DNA flow cytometry.
DNA was stained with propidium iodide (PI), and mitotic cells were
quantified by measuring the expression of the mitosis-specific
marker phospho-histone H3, as previously described.[47] Phospho-
histone H3 levels and the DNA content of individual cells were an-
alyzed using a fluorescence-activated cell sorter (EpicsXL/MCL,
2-Phenylquinolin-4-yl-4-nitrobenzenesulfonate (10m): Com-
pound 10m was prepared from 2-phenylquinolin-4(1H)-one (13j,
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L.-H. Yih, T.-L. Su, et al.
MED
Beckman Coulter, Fullerton, CA), and the cell cycle distribution of
the cells was determined using EXPO32 ADC Analysis software pro-
vided by Beckman Coulter.
Colchicine competitive binding assay
The ability of the compounds to compete with colchicine for bind-
ing to tubulin was assessed by measuring the change in fluores-
cence of the tubulin–colchicine complex.[34] Tubulin (5 mm) was
pre-incubated with 20 mm quinoline sulfonates, 20 mm colcemid
(Sigma), or 20 mm vinblastine (Sigma) for 45 min at 378C. Next,
20 mm colchicine (Sigma) was added to all reaction mixtures. Spec-
tra were recorded after 20 min incubation by exciting the samples
at 360 nm and measuring emission at 435 nm using a spectro-
fluorimeter (SpectraMax M5, Molecular Devices).
Detection of apoptosis
Apoptotic cells were identified by flow cytometry analysis of the
level of cleaved poly(ADP-ribose) polymerase (PARP).[48] Briefly, cells
were fixed with ice-cold 70% EtOH for 16 h, then immunostained
for 3 h at room temperature with rabbit anti-human cleaved PARP
antibodies (Cell Signaling Technology, Beverly, MA), followed by in-
cubation for 1 h at room temperature with allophycocyanin-conju-
gated goat anti-rabbit IgG (Invitrogen). Cellular DNA was counter-
stained with PI. Levels of cleaved PARP and PI in individual cells
were analyzed using a fluorescence-activated cell sorter, and the
percentage of cells with cleaved PARP was determined using
EXPO32 ADC Analysis software provided by Beckman Coulter.
BODIPY FL-vinblastine competitive binding assay
The ability of the compounds to compete with vinblastine for
binding to tubulin was assessed by measuring the change in fluo-
rescence of the BODIPY FL-vinblastine–tubulin complex. Tubulin
(3 mm) was pre-incubated with either 20 mm quinoline sulfonate or
20 mm vinblastine for 45 min in buffer containing 80 mm PIPES
pH 6.9, 0.5 mm EGTA and 2 mm MgCl2, followed by the addition of
BODIPY FL-vinblastine (3 mm, Invitrogen, Carlsbad, CA). After
20 min incubation, the samples were excited at 490 nm and emis-
sion spectra were monitored from 505 nm to 530 nm using a spec-
trofluorimeter (SpectraMax M5, Molecular Devices).
Immunofluorescence staining
Cells seeded on glass cover slips were incubated for 14 h at 378C
with or without drugs, washed twice with phosphate-buffered
saline (PBS), and fixed in situ with 90% MeOH at À208C for 10 min.
The cells were then immunostained for mitotic spindles with an
anti-human a-tubulin antibody (Sigma, St. Louis, MO, USA) and for
centrosomes with an anti-human g-tubulin antibody (Sigma) as
previously described.[47] Nuclei were simultaneously counterstained
with 0.1 mgmLÀ1 of 4,6-diamino-2-phenylindole (DAPI, Sigma). After
thorough rinsing with PBS containing 0.2% (v/v) Tween 20, the
cells were mounted using a 90% glycerol solution and examined
under a fluorescence microscope (Axioskop 2, Zeiss, Oberkochen,
Germany).
Abbreviations
CA-4, combretastatin A-4; CCRF-CEM, human lymphoblastic
leukemia; DAPI, 4,6-diamino-2-phenylindole; DMF, N,N-dime-
thylformamide; HCT-116, human colon carcinoma; HeLa-S3,
human epithelial carcinoma; H460, human large-cell lung carci-
noma; HT29, human colon adenocarcinoma; MDR, multidrug
resistance; PARP, poly(ADP-ribose) polymerase; PBS, phos-
phate-buffered saline; PC-3, prostate adenocarcinoma; PI, pro-
pidium iodide; SAR, structure–activity relationship.
Cellular microtubule stabilization assay
The effect of sulfonate derivatives on microtubule stabilization was
assessed using the cellular microtubule stabilization assay.[49] After
treatment with the indicated compounds, HeLa-S3 cells were har-
vested in lysis buffer (100 mm PIPES pH 6.9, 1 mm EGTA, 1 mm
MgCl2, 30% glycerol, 0.5% DMSO, 1% NP-40, 1 mm GTP, and pro-
tease inhibitors). Following centrifugation at 180000 g at 378C for
1 h, the polymerized tubulin fraction (pellet) and the soluble tubu-
lin fraction (supernatant) were separated. These two fractions were
adjusted to the same volume with Laemmli sample buffer. Finally,
the amount of a-tubulin from equal aliquots of the polymerized
tubulin fraction and the soluble tubulin fraction was determined
by immunoblot analyses. Mitotic cells, released for 10 h from a
double-thymidine block, were used as a control.
Acknowledgements
We are grateful for support from the National Science Council,
Taiwan, Republic of China (NSC 98-2320-B-001-005 to T.-L.S. and
NSC 99-2320-B-001-008-MY3 to L.-H.Y.). T.-C.C. was supported by
the Sloan-Kettering Institute General Fund. NMR spectra of syn-
thesized compounds were obtained at the High-Field Biomolecu-
lar NMR Core Facility supported by the National Research Pro-
gram for Genomic Medicine (Taiwan). We thank Dr. Shu-Chuan
Jao at the Institute of Biological Chemistry (Academia Sinica) for
providing NMR service and the National Center for High-Perfor-
mance Computing for computer time and facilities.
Keywords: antitumor agents · cytotoxicity · mitotic arrest ·
sulfonates · tubulin polymerization
Determination of intrinsic tryptophan fluorescence of tubulin
Intrinsic tryptophan fluorescence of tubulin was used to measure
the binding affinity of drugs to tubulin.[50] Porcine brain tubulin
(3 mm, Cytoskeleton Inc., Denver, CO, USA) was incubated with the
indicated drugs at 378C for 40 min in buffer containing 80 mm
PIPES pH 6.9, 0.5 mm EGTA, and 2 mm MgCl2. The samples were
then excited at 295 nm, and the emission spectrum was monitored
from 325 nm to 370 nm with a spectrofluorimeter (SpectraMax M5,
Molecular Devices, Sunnyvale, CA, USA).
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Received: March 1, 2011
Revised: April 1, 2011
Published online on May 3, 2011
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1129