Design, synthesis, and biological evaluation of novel N-γ-carboline arylsulfonamides as anticancer agents

Article abstract of DOI:10.1016/j.bmc.2010.10.047

A series of novel N-γ-carboline arylsulfonamide derivatives designed based on the common feature of colchicine binding site inhibitors were synthesized and evaluated for their antiproliferative activity in vitro against five human cancer cell lines. Most

Full text of DOI:10.1016/j.bmc.2010.10.047

Bioorganic & Medicinal Chemistry 18 (2010) 8478–8484
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of novel N-c-carboline
arylsulfonamides as anticancer agents
Jing Chen a,b, Tao Liu a, Rui Wu c, Jianshu Lou c, Ji Cao c, Xiaowu Dong a, Bo Yang c, Qiaojun He c,
Yongzhou Hu a,
⇑
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058 Zhejiang, China
b College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053 Zhejiang, China
c Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058 Zhejiang, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel N-c-carboline arylsulfonamide derivatives designed based on the common feature of
Received 28 August 2010
Revised 16 October 2010
Accepted 19 October 2010
Available online 25 October 2010
colchicine binding site inhibitors were synthesized and evaluated for their antiproliferative activity
in vitro against five human cancer cell lines. Most of the compounds showed moderate to potent cyto-
toxic activities against all the tested cells. Preliminary mechanism research on one of the most potent
compound 6p indicated that it was a potent tubulin polymerization inhibitor, with IC50 value of
3.8
l
M, equivalent to that of CA-4, and arresting cell cycle in G2/M phase.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
N-c-Carboline arylsulfonamides
Anticancer
Antitubulin
DNA intercalation
1. Introduction
and hydrophobic groups or polar functional groups at ring B are
beneficial for binding affinity.
Antimitotic agents, which arrest the cell cycle at the G2/M phase
and lead to apoptotic cell death and tumor regression, have been
the most successful pharmacological agents for the treatment of
cancer.1,2 Most of the antimitotics are tubulin-binding agents. So
far, several small-molecule antimitotics have been developed to
antagonize one of the three major binding-sites on tubulin,
namely, the vinca-, the taxus-, and the colchicine binding site.3
While the vinca alkaloids and the taxanes have well-established
roles in the treatment of human cancers,4–6 no colchicine binding
site inhibitors (CSIs) are currently approved for cancer therapy.
However, a large number of natural and synthetic small molecules
of varied structures have been identified as CSIs over the years,7,8
some are undergoing clinical trials for cancer therapy, such as com-
bretastatin A-4 phosphate (CA-4P, 1) and E7010 (2),9,10 thus sug-
gesting a high plasticity of this binding site. Quite a few attempts
to describe the common pharmacophore and structural features
of CSIs have been made,11,12 which can be used for rational design
of new antitubulin agents. It is reported that most CSIs bear two
hydrophobic groups (ring A and B) and a cis-bridge between them.
The volume size of ring A is often bigger than ring B. It is also re-
ported that the existence of hydrogen-bond acceptor at ring A
2. Design
During our studies on new antitumor agents, we previously re-
ported three series of potent cytotoxic compounds, characterized
by the presence of
c-carboline connected with aromatic rings
through different linkers (3–5, Fig. 1).13 We supposed that these
O2
S
N
N
H
A
B
H3CO
OP(O)(ONa)2
NH
H3CO
H3CO OCH3 OCH3
HO
1: CA-4P
2: E7010
X
linker
O2
R2
S
HN
N
hydrogen-bond
acceptor
Ar
ring B
N
N
R1
N
R1
3: X=SO2NH
4: SO3,
5: NHCO
ring A
6
⇑
Corresponding author. Tel./fax: +86 571 88208460.
E-mail addresses: huyz@zjuem.zju.edu.cn, huyz@zju.edu.cn (Y. Hu).
Figure 1. Structure of CA-4P, E7010, and c-carboline derivatives.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.047

J. Chen et al. / Bioorg. Med. Chem. 18 (2010) 8478–8484
8479
compounds match well with the common structure of CSIs: the
human gastric adenocarcinoma SGC, human colon cancer cell
HCT116, human breast carcinoma cell MCF-7, and human myeloid
leukemia cell K562 by MTT assay for 48 h. Taxol and previously re-
ported compounds 3a–c was employed as the positive control. The
results are summarized in Table 2.
As shown in Table 2, most of the tested compounds showed
moderate to potent cytotoxic activities against all the tested cells.
Ten of the twenty-six new compounds showed strong cytotoxic
planar c-carboline acts as the hydrophobic ring A of CSIs, and the
N2 position of carboline contributes as a hydrogen-bond acceptor;
the aromatic ring connected to the linker is the hydrophobic ring B
of CSIs. Further mechanism study showed that these compounds
exhibited antitubulin activity (data haven’t been revealed yet).
Enlightened by the results and for the purpose of further study
on structure–activity relationships (SARs) of
as developing more potent antitubulin agents, we reversed the lin-
ker of compounds 3 and designed a novel series of N- -carboline
c-carbolines, as well
activity against more than three cell lines (IC50 <10 lM). Among
them, compounds 6p and 6t were the most promising compounds
c
arylsulfonamide derivatives 6. According to the reported feature
of CSIs,12 we also introduced hydrophobic substituents, such as
halogen and methoxy group, as well as polar functional groups,
such as amino group, on ring B (Fig. 1). We calculated ‘cdocker-en-
ergy’ of 3a–c and 6a–c by molecular modeling studies using the
Discovery Studio 2.1/cdocker approach. The results showed that
the ‘cdocker-energy’ of the new designed compounds 6a–c were
lower than those of the corresponding compounds 3a–c except
for compound 6b (Table 1). This result implied that reversing the
linker of compounds 3 might lead to the more effective antitubulin
agents. Besides, carboline skeleton has been identified as an excel-
lent scaffold structure with DNA intercalation.14 Thus, we expected
the resulting compounds would possess improved antitumor activ-
ities by interaction with both tubulin and DNA. Here, we report the
against all the tested cell lines, with IC50 values of 0.47–11.29 and
0.17–6.73
The N5-phenylsulfonyl substituted compounds 6n and 6o
exhibited no activities against all the tested cells (IC50 >100 M),
lM, respectively.
l
except for K562 cell. N5-Boc or N5-ethyl substituted compounds
were more potent cytotoxic activity than those of N5 unsubstituted
compounds (6z vs 6g, 6p vs 6c, 6r vs 6i). These implied that intro-
ducing proper substituents at N5 position was beneficial to the
cytotoxic activity. In addition, compound 6y, with an ethyl group
at the nitrogen atom of sulfonamide group, showed significantly
increased potency (fivefold) against K562 cell in comparison with
6p. This indicated that introducing ethyl group to the linker was
beneficial to cytotoxic activity. This result was similar to the SAR
of our previously reported series 3.13
synthesis and biological evaluation of a series of novel N-
line arylsulfonamides. The docking studies of the interaction will
also be discussed.
c
-carbo-
The influence of various substituents on the phenyl ring was
also examined. Comparing the cytotoxic activities of compounds
6c–e revealed that the effects of different halogen atom at para-po-
sition of phenyl ring was: chlorine > bromine > fluorine atom.
However, two chlorine atoms substituted compound 6f exhibited
decreased activity. Besides, a comparison between compounds 6q
and 6s–v revealed the influence of methoxy group at different
position of phenyl ring. It could be concluded as: 2,4-dime-
thoxy > 3,4-dimethoxy > 4-methoxy > 2,4,6-trimethoxy > 2,5-dime-
thoxy. Moreover, replacement of the phenyl with naphthalene led
compound 6m, which retained potent cytotoxic activity against
3. Chemistry
The synthetic routes of N-
tives 6a–z were diagrammed in Scheme 1.13
pared using our recently developed synthetic protocol.15 Compound
7 was nitrified with concd HNO3–H2SO4 to provide 6-nitro- -carbo-
c-carboline arylsulfonamide deriva-
c-Carboline 7 was pre-
c
line 8a, and then the N5 of 8a was protected as carbamate 8b or alkyl-
ated with C2H5Br to give compound 8c. Reduction the nitro group of
compound 8 with Raney Ni in EtOH yielded the corresponding ami-
no compound 9a–c. Condensation of compound 9a or 9c with corre-
sponding arylsulfonyl chlorides in the presence of pyridine in DMF
at room temperature afforded the N5 substituted products 6n–w.
Among them, the 30-nitro group of compound 6r could be reduced
with Raney Ni to yield the 30-amino compound 6x, while compound
6p was further transformed into 6y by the N-alkylation of sulfon-
amide group with C2H5Br. The N5 unsubstituted arysulfonamides
6a–m were obtained through two steps. First, reaction of 9b with
various arylsulfonyl chlorides to give the N5-tert-butoxycarbonyl
(Boc) substituted compounds, such as 6z. Then, N5-Boc substituted
compounds could be transformed into the target compounds 6a–
m by hydrolysis with concd HCl–EtOH.
A549 and SGC cell lines, with IC50 values of 8.80 and 5.65
respectively.
At last, it was worthy to point out that the cytotoxic activity of
compounds 6a–c were more potent than our previously synthe-
sized compounds 3a–c (Table 2).
Because the compounds were designed to binding to tubulin,
flow cytometry experiments were carried out to determine
whether the synthesized compounds could lead to cell cycle arrest
lM,
at G2/M phase. A549 cells were treated with 6p (2
lM), one of the
most potent compounds, for 48 h, and CA-4 (1 M) was used as a
l
positive control. Figure 2 showed that compound 6p caused a
marked increase in the percentage of cells in the G2/M phase of
the cell cycle, with a simultaneous decrease of cells in G0/G1 and
S phase. It was in the similar trend as CA-4.
To confirm whether tubulin was the target of these compounds,
five of the most promising N-c-carboline arylsulfonamides 6p, 6t–
4. Biological studies and discussion
v, and 6y were evaluated for their inhibition of polymerization of
purified tubulin in a cell-free system. CA-4 was used as a positive
control. The results are shown in Figure 3. In this assay, CA-4 inhib-
The synthesized N-c-carboline arylsulfonamides 6 were tested
for their cytotoxic activities in vitro against several human cancer
ited tubulin polymerization by 85.7% at 3
lM. All the tested
cell lines including human non-small lung cancer cells A549,
compounds (6p, 6t–v and 6y) displayed a pattern similar to CA-4,
Table 1
Calculated ‘cdocker-energy’ of compounds 3a–c and 6a–c
No.
Structure
R
Cdocker-energy
No.
Structure
R
Cdocker-energy
3a
3b
3c
H
OCH3
Br
ꢀ14.345
ꢀ17.466
6a
6b
6c
H
OCH3
Br
ꢀ18.749
ꢀ17.149
NH
SO2
HN
O2S
ꢀ17.611
ꢀ18.656
N
N
R
R
N
H
N
H

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J. Chen et al. / Bioorg. Med. Chem. 18 (2010) 8478–8484
SO2
HN
N
CH3
6a: Ar=Ph
N
6b: Ar=4-CH3O-Ph
6c: Ar=4-Br-Ph
6d: Ar=4-Cl-Ph
6e: Ar=4-F-Ph
6f: Ar=2, 5-diCl-Ph
6g: Ar=4-CH3-Ph
Boc
6z
SO2
Ar
HN
NO2
NH2
e
d
N
N
N
6h: Ar=3-NO2-4-CH3O-Ph
6i: Ar=3-NO2-Ph
6j: Ar=2, 5-diCH3O-Ph
6k: Ar=4-CF3O-Ph
e, f
N
H
N
N
Boc
9b
Boc
6a-m
8b
6l: Ar=2, 4, 6-triCH3-Ph
b
SO2
6m: Ar=
HN
OCH3
NO2
NH2
Ar
a
e
d
N
N
N
N
N
H
N
N
H
N
O2S
H
Ar
6n, 6o
6n: Ar=4-Br-Ph
6o: Ar=4-CH3-Ph
7
8a
9a
c
SO2
Ar
NH2
HN
NO2
6p: Ar=4-Br-Ph
6q: Ar=4-CH3O-Ph
6r: Ar=3-NO2-Ph
e
d
N
N
N
N
N
N
C2H5
C2H5
C2H5
6s: Ar=2, 5-diCH3O-Ph
6t: Ar=2, 4-diCH3O-Ph
6u: Ar=3, 4-diCH3O-Ph
6v: Ar=2, 4, 6-triCH3O-Ph
6w: Ar=4-PhO-Ph
6x: Ar=3-NH2-Ph
9c
6p-x
8c
d
c
C2H5
SO2
Ar
N
6y: Ar=4-Br-Ph
N
N
C2H5
6y
Scheme 1. Reagents and conditions: (a) HNO3–H2SO4, 0 °C to rt, 4 h; (b) (Boc)2O, DMAP, dry THF, N2, rt 24 h; (c) C2H5Br, NaH, dry THF–DMF, rt, 0.5 h; (d) Raney Ni, EtOH, H2,
rt, 2 h; (e) ArSO2Cl, pyridine, DMF, rt, 0.5 h; (f) concd HCl–EtOH, rt, 12 h.
Table 2
In vitro cytotoxic activities of the synthesized compounds against five human cancer cell lines
Compd
Cytotoxicity (IC50
HCT116
,
l
M)a,b
MCF7
Compd
Cytotoxicity (IC50
, lM)
A549
SGC
K562
A549
SGC
1.51
HCT116
MCF7
K562
c
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
45.52
23.68
9.62
5.25
6.65
60.29
16.83
38.44
38.44
17.11
7.07
91.29
11.69
40.17
4.67
—
61.97
17.94
7.91
14.16
1.39
5.97
3.47
1.64
16.70
19.62
28.91
18.31
5.84
2.00
6p
6q
6r
6s
6t
6u
6v
6w
6x
6y
6z
3a
3b
3c
1.88
15.21
10.62
44.26
3.50
10.81
21.00
11.45
—
7.78
21.05
78.39
—
11.29
9.68
6.38
37.70
6.00
12.76
21.20
21.85
—
3.58
0.89
35.16
—
3.79
3.70
1.31
2.62
6.73
11.15
6.34
9.46
—
0.47
3.75
7.67
3.01
0.17
0.12
0.068
0.65
9.59
11.91
—
15.63
8.30
17.59
0.29
0.41
2.20
5.08
6.74
2.11
42.35
>100
—
19.00
>100
76.22
10.85
30.25
31.71
15.78
11.31
27.45
18.02
—
8.41
58.24
—
57.73
—
48.79
49.35
21.15
24.94
55.06
6.06
—
0.82
3.49
27.79
>100
16.83
>100
>100
1.35
—
0.094
5.51
7.69
8.80
>100
>100
18.96
5.65
2.33
—
40.93
35.40
60.12
—
—
21.03
33.87
16.43
1.16
>100
>100
—
2.46
—
3.34
—
4.37
—
Taxol
2.11
a
b
c
IC50, compound concentration required to inhibit tumor cell proliferation by 50%.
Values are means of three experiments.
NT, not tested.
and they inhibited tubulin polymerization by 60.6%, 43.4%, 37.7%,
35.1%, and 30.6%, respectively, at 4 M. The most potent antitubu-
lin compound 6p was further examined at concentrations from 1 to
8
3.8
l
M. The IC50 value of its tubulin polymerization inhibition was
M, similar to that of CA-4 (1.5 M). Comparing the tubulin
inhibition with corresponding cytotoxic activities revealed that
l
l
l

J. Chen et al. / Bioorg. Med. Chem. 18 (2010) 8478–8484
8481
Figure 2. Effect of compound 6p (2 lM) and CA-4 (1 lM) on the cell cycle of A549 cells.
Figure 3. Effect of compounds 6p, 6t–v, and 6y (4 lM) on tubulin assembly.
Figure 5. Proposed binding mode for CA-4 (in green), compounds 6p (in blue) and
6y (in mauve) in the colchicine site of tubulin.
the two data were correlated except for compound 6y, which
showed increased potency (fivefold) against K562 cell with half de-
creased tubulin inhibition compared with 6p. It implied that com-
pound 6y might act against another target.
In view of compounds possessing carboline moiety displayed
DNA intercalation.14 Compounds 6p, 6t, and 6y were also evalu-
ated by DNA interaction assay (ethidium bromide (EB) displace-
ment) (Fig. 4). It showed that the fluorescence of the DNA-EB
complex was diminished only by treatment with compound 6y,
while compounds 6p and 6t had no effect on it. This result indi-
nism of cytotoxic activity of 6y was different from the other syn-
thesized N-c-carboline arylsulfonamides.
The proposed mechanism of action was also supported by dock-
ing studies of compounds 6p and 6y in the colchicine site of tubu-
lin by using the reported high-resolution crystal structure of
tubulin-DAMAcolchicine complex.16 Figure 5 showed both com-
pounds 6p and 6y located in the same pocket on b-tubulin as
CA-4. Among them, the docking conformation of compound 6p
cated that most of the N-c-carboline arylsulfonamides couldn’t
intercalate DNA. But introducing ethyl group at the nitrogen atom
of sulfonamide group would improve its DNA affinity. Combined
with the weak tubulin inhibition of 6y, we supposed the mecha-
well overlaps with CA-4 in the crystallized protein complex.
Carboline moiety and the substituted phenyl ring lay in the hydro-
phobic pocket between Alab250–Alab316 and Val 181–Metb259,
c-
a
respectively. Introduction of ethyl group at the nitrogen atom of
sulfonamide group (6y) would lead to the modification of the con-
formation as shown in Figure 5.
c-Carboline moiety shift away
form the hydrophobic pocket. This result was in accordance with
the observation that compound 6y is not as effective as compound
6p as antitubulin agent. Additionally, N2 position of carboline
formed a hydrogen-bond with Cysb241, which always found in
CSIs.
5. Conclusion
In summary, a series of novel N-c-carboline arylsulfonamides
were synthesized by reverse the sulfonamide linkage of the lead
compounds 3. Most of the synthesized compounds showed moder-
ate to potent cytotoxic activities against all the tested cells, and im-
proved activity than the lead compounds 3. Preliminary
mechanism research indicated the synthesized compounds could
inhibit tubulin polymerization. The IC50 value of compound 6p
Figure 4. In vitro DNA intercalation assay for compounds 6p, 6t and 6y (0.2 lM).

8482
J. Chen et al. / Bioorg. Med. Chem. 18 (2010) 8478–8484
was 3.8
l
M, similar to that of CA-4. Moreover, it could also lead to
(d, 1H, J = 6.4 Hz), 8.09 (s, 1H), 7.74 (m, 3H), 7.63 (d, 2H,
J = 8.0 Hz), 7.57 (d, 1H, J = 8.0 Hz), 7.23 (d, 1H, J = 8.0 Hz). ESI-MS:
m/z = 402 [M+1]+. Anal. Calcd for C17H12BrN3O2S: C, 50.76; H,
3.01; N, 10.45. Found: C, 50.75; H, 2.95; N, 10.34.
cell cycle arrest at G2/M phase. Introducing ethyl group at the
nitrogen atom of sulfonamide group gave more cytotoxic com-
pound 6y with reduced antitubulin activity. Further DNA interac-
tion assay indicated this modification would improve its DNA
affinity. With all these results, further design and synthesis of po-
tent antitumor agents are ongoing in our laboratory and the results
will be reported in due course.
6.1.1.4. 4-Chloro-N-(5H-c-carboline-8-yl)benzenesulfonamide
(6d). White solid (36%), mp: 119–121 °C. 1H NMR (d, DMSO-d6):
11.76 (br s, 1H, NH), 10.21 (br s, 1H, NH), 9.26 (s, 1H), 8.40
(d, 1H, J = 3.6 Hz), 7.79 (s, 1H), 7.70 (d, 2H, J = 8.0 Hz), 7.59 (d, 2H,
J = 8.0 Hz), 7.44 (m, 2H), 7.12 (dd, 1H, J = 8.0, 1.6 Hz). ESI-MS: m/
z = 358 [M+1]+. Anal. Calcd for C17H12ClN3O2S: C, 57.06; H, 3.38;
N, 11.74. Found: C, 56.94; H, 3.52; N, 11.58.
6. Experimental
Reagents and solvents were purchased from commercial
sources. Melting points were obtained on a B-540 Büchi melting-
point apparatus and are uncorrected. 1H NMR spectra was re-
corded on a Brüker AM 400 instrument at 400 MHz (chemical
shifts are expressed as d values relative to TMS as internal stan-
dard). ESI (positive) was recorded on an Esquire-LC-00075 spec-
trometer. Element analyses were performed on an Eager 300
instrument.
6.1.1.5. 4-Fluoro-N-(5H-c-carboline-8-yl)benzenesulfonamide
(6e). White solid (70%), mp: 151–153 °C. 1H NMR (d, DMSO-d6):
11.68 (br s, 1H, NH), 10.16 (br s, 1H, NH), 9.24 (s, 1H), 8.39
(d, 1H, J = 6.0 Hz), 7.89 (s, 1H), 7.76 (m, 2H), 7.42 (m, 2H), 7.35
(t, 2H, J = 8.0 Hz), 7.11 (d, 1H, J = 8.0 Hz). ESI-MS: m/z = 342
[M+1]+. Anal. Calcd for C17H12FN3O2S: C, 59.81; H, 3.54; N, 12.31.
Found: C, 59.87; H, 3.76; N, 12.45.
6.1. Synthesis
6.1.1.6.
2,5-Dichloro-N-(5H-c-carboline-8-yl)benzenesulfon-
amide (6f). White solid (43%), mp: >250 °C. 1H NMR (d, DMSO-
d6): 11.17 (br s, 1H, NH), 9.01 (s, 1H), 8.26 (d, 1H, J = 5.2 Hz), 7.93
(d, 1H, J = 2.0 Hz), 7.54 (s, 1H), 7.40 (d, 1H, J = 8.8 Hz), 7.34 (dd,
1H, J = 8.4, 2.0 Hz), 7.29 (d, 1H, J = 5.2 Hz), 7.16 (d, 1H, J = 8.4 Hz),
6.99 (d, 1H, J = 8.8 Hz). ESI-MS: m/z = 392 [M+1]+. Anal. Calcd for
6.1.1. General procedure for synthesis of N-c-carboline
arylsulfonamides 6
A mixture of 5-ethyl-8-amino-
sulfonyl chlorides (1 mmol) in DMF (5 mL) was stirred for 5 min at
room temperature, pyridine (324 L, 4 mmol) was added. Then,
c-carboline 9c (1 mmol), aryl-
l
the mixture was stirred for an additional 0.5 h. After adding ice
water (10 mL), the mixture was extracted with EtOAc (3 ꢁ 20 mL).
The organic phase was washed with brine (2 ꢁ 20 mL), dried over
anhydrous Na2SO4, and concentrated under vacuum. The residue
was purified over silica column chromatography using petroleum
ether (PE)/EtOAc/EtOH (20:20:1, v/v/v) as eluent to afford N5-ethyl
products 6p–w.
C
17H11Cl2N3O2S: C, 52.05; H, 2.83; N, 10.71. Found: C, 51.97; H,
2.57; N, 10.43.
6.1.1.7. 4-Methyl-N-(5H-c-carboline-8-yl)benzenesulfonamide
(6g). White solid (57%), mp: 115–117 °C. 1H NMR (d, DMSO-d6):
11.66 (br s, 1H, NH), 9.20 (s, 1H), 8.37 (d, 1H, J = 5.6 Hz), 7.85
(d, 1H, J = 1.6 Hz, H-5), 7.60 (d, 2H, J = 8.0 Hz), 7.41 (d, 1H,
J = 5.6 Hz), 7.34 (d, 1H, J = 8.8 Hz), 7.27 (d, 2H, J = 8.0 Hz), 7.12
(dd, 1H, J = 8.8, 1.6 Hz), 2.2 (s, 3H). ESI-MS: m/z = 338 [M+1]+. Anal.
Calcd for C18H15N3O2S: C, 64.08; H, 4.48; N, 12.45. Found: C, 64.24;
H, 4.64; N, 12.14.
In the same manner, the N5-phenylsulfonyl products 6n and 6o
were synthesized by reaction of 8-amino-
with corresponding arylsulfonyl chlorides (2 mmol). N5-Boc prod-
uct (e.g., 6z) were synthesized by reaction of 5-Boc-8-amino-
c-carboline 9a (1 mmol)
c
-
carboline 9b (1 mmol) with various arylsulfonyl chloride (1 mmol).
Then the N5-Boc product (0.5 mmol) was mixed with concd HCl–
EtOH (1 mL, 1:1), and stirred overnight at room temperature. After
that, the mixture was filtrated. The residue was neutralized with
10% NaOH, and extracted with AcOEt (3 ꢁ 5 mL). The organic phase
was washed with brine (2 ꢁ 5 mL), dried over anhydrous Na2SO4,
and concentrated under vacuum to afford N5-unsubstituted prod-
ucts 6a–m.
6.1.1.8. 4-Methoxy-3-nitro-N-(5H-c-carboline-8-yl)benzenesul-
fonamide (6h). White solid (60%), mp: 118–120 °C. 1H NMR
(d, DMSO-d6): 11.88 (br s, 1H, NH), 10.26 (br s, 1H, NH), 9.35
(s, 1H), 8.45 (d, 1H, J = 4.8 Hz), 8.24 (s, 1H), 8.00 (s, 1H), 7.92
(d, 1H, J = 8.8 Hz), 7.51 (d, 1H, J = 4.8 Hz), 7.50 (d, 2H, J = 8.8 Hz),
7.18 (d, 1H, J = 8.8 Hz), 3.96 (s, 3H). ESI-MS: m/z = 399 [M+1]+. Anal.
Calcd for C16H14N4O5S: C, 54.27; H, 3.54; N, 14.06. Found: C, 54.52;
H, 3.35; N, 14.36.
6.1.1.1. N-(5H-c-Carboline-8-yl)benzenesulfonamide (6a). White
solid (78%), mp: 217–219 °C. 1H NMR (d, DMSO-d6): 11.63 (br s, 1H,
NH), 9.18 (s, 1H), 8.37 (d, 1H, J = 5.2 Hz), 7.83 (s, 1H), 7.72 (d, 2H,
J = 8.0 Hz), 7.51 (m, 3H), 7.39 (d, 1H, J = 5.2 Hz), 7.36 (d, 1H,
J = 8.4 Hz), 7.10 (d, 1H, J = 8.4 Hz). ESI-MS: m/z = 324 [M+1]+. Anal.
Calcd for C17H13N3O2S: C, 63.14; H, 4.05; N, 12.99. Found: C, 63.26;
H, 3.85; N, 12.88.
6.1.1.9.
3-Nitro-N-(5H-c-carboline-8-yl)benzenesulfonamide
(6i). White solid (37%), mp: 189–191 °C. 1H NMR (d, DMSO-d6):
11.93 (br s, 1H, NH), 10.50 (br s, 1H, NH), 9.35 (s, 1H), 8.50
(s, 1H), 8.45 (m, 2H), 8.08 (d, 1H, J = 8.0 Hz), 7.99 (s, 1H), 7.83 (t,
1H, J = 8.0 Hz), 7.52 (d, 1H, J = 5.6 Hz), 7.49 (d, 1H, J = 8.0 Hz),
7.17 (dd, 1H, J = 8.0, 2,0 Hz). ESI-MS: m/z = 369 [M+1]+. Anal. Calcd
for C17H12N4O4S: C, 55.43; H, 3.28; N, 15.21. Found: C, 55.62; H,
3.32; N, 15.43.
6.1.1.2. 4-Methoxy-N-(5H-c-carboline-8-yl)benzenesulfonamide
(6b). White solid (81%), mp: 115–117 °C. 1H NMR (d, DMSO-d6):
12.39 (br s, 1H, NH), 10.11 (br s, 1H, NH), 9.52 (s, 1H), 8.50 (d, 1H,
J = 5.2 Hz), 8.06 (d, 1H, J = 2.0 Hz), 7.68 (m, 3H), 7.53 (d, 1H,
J = 8.8 Hz), 7.23 (dd, 1H, J = 8.8, 2.0 Hz), 7.02 (d, 2H, J = 8.8 Hz),
3.75 (s, 3H). ESI-MS: m/z = 354 [M+1]+. Anal. Calcd for C16H15N3O3S:
C, 61.18; H, 4.28; N, 11.89. Found: C, 61.24; H, 4.52; N, 12.11.
6.1.1.10. 2,5-Dimethoxy-N-(5H-c-carboline-8-yl)benzenesulfon-
amide (6j). White solid (63%), mp: 187–189 °C. 1H NMR
(d, DMSO-d6): 12.77 (br s, 1H, NH), 10.03 (br s, 1H, NH), 9.63 (s,
1H), 8.52 (d, 1H, J = 6.0 Hz), 8.09 (s, 1H), 7.77 (d, 1H, J = 6.0 Hz),
7.57 (d, 1H, J = 8.0 Hz), 7.33 (dd, 1H, J = 8.0, 1.6 Hz), 7.22 (d, 1H,
J = 2.8 Hz), 7.12 (m, 2H), 3.87 (s, 3H), 3.65 (s, 3H). ESI-MS: m/
z = 384 [M+1]+. Anal. Calcd for C19H17N3O4S: C, 59.52; H, 4.47; N,
10.96. Found: C, 59.53; H, 4.68; N, 11.02.
6.1.1.3. 4-Bromo-N-(5H-c-carboline-8-yl)benzenesulfonamide
(6c). White solid (54%), mp: 183–185 °C. 1H NMR (d, DMSO-d6):
12.59 (br s, 1H, NH), 10.38 (br s, 1H, NH), 9.58 (s, 1H), 8.52

J. Chen et al. / Bioorg. Med. Chem. 18 (2010) 8478–8484
8483
6.1.1.11. 4-Trifluoromethoxy-N-(5H-
c
-carboline-8-yl)benzene-
J = 7.2 Hz), 3.71 (q, 2H, J = 7.2 Hz), 1.34 (t, 3H, J = 7.2 Hz), 1.03
(t, 3H, J = 7.2 Hz). ESI-MS: m/z = 458 [M+1]+. Anal. Calcd for
sulfonamide (6k). White solid (81%), mp: 246–248 °C. 1H NMR
(d, DMSO-d6): 12.07 (br s, 1H, NH), 10.30 (br s, 1H, NH), 9.37
(s, 1H), 8.44 (d, 1H, J = 4.4 Hz), 7.98 (s, 1H), 7.83 (d, 2H,
J = 8.4 Hz), 7.55 (m, 4H), 7.16 (d, 1H, J = 8.4 Hz). ESI-MS: m/z =
408 [M+1]+. Anal. Calcd for C16H12F3N3O3S: C, 53.07; H, 2.97; N,
10.32. Found: C, 53.24; H, 2.79; N, 10.42.
C21H20BrN3O2S: C, 55.03; H, 4.40; N, 9.17. Found: C, 54.89; H,
4.24; N, 9.15.
6.1.1.17. 4-Methoxy-N-(5-ethyl-5H-c-carboline-8-yl)benzene-
sulfonamide (6q). White solid (40%), mp: 122–124 °C. 1H NMR
(d, DMSO-d6): 9.24 (s, 1H), 8.45 (d, 1H, J = 6.0 Hz), 7.93 (d, 1H,
J = 2.0 Hz), 7.67 (d, 2H, J = 8.8 Hz), 7.59 (d, 1H, J = 6.0 Hz), 7.56
(d, 1H, J = 8.8 Hz), 7.21 (dd, 1H, J = 2.0, 8.8 Hz), 7.01 (d, 2H,
J = 8.8 Hz), 4.38 (q, 2H, J = 7.2 Hz), 3.75 (s, 3H), 1.28 (t, 3H,
J = 7.2 Hz). ESI-MS: m/z = 382 [M+1]+. Anal. Calcd for C20H19N3O3S:
C, 62.97; H, 5.02; N, 11.02. Found: C, 63.14; H, 5.35; N, 11.25.
6.1.1.12.
2,4,6-Trimethyl-N-(5H-c-carboline-8-yl)benzenesul-
fonamide (6l). White solid (30%), mp: >250 °C. 1H NMR
(d, DMSO-d6): 11.65 (br s, 1H, NH), 9.15 (s, 1H), 8.36 (d, 1H,
J = 5.2 Hz), 7.75 (s, 1H), 7.40 (d, 1H, J = 5.2 Hz), 7.30 (d, 1H,
J = 8.8 Hz), 7.04 (d, 1H, J = 8.8 Hz), 6.91 (s, 2H), 2.17 (s, 9H). ESI-
MS: m/z = 366 [M+1]+. Anal. Calcd for C20H19N3O2S: C, 65.73; H,
5.24; N, 11.50. Found: C, 65.57; H, 5.36; N, 11.64.
6.1.1.18.
3-Nitro-N-(5-ethyl-5H-c-carboline-8-yl)benzenesul-
fonamide (6r). White solid (44%), mp: 159–161 °C. 1H NMR
(d, DMSO-d6): 10.46 (br s, 1H, NH), 9.33 (s, 1H), 8.49 (d, 2H,
J = 1.2 Hz), 8.44 (dd, 1H, J = 1.2, 8,8 Hz), 8.08 (d, 1H, J = 8.8 Hz),
8.00 (d, 1H, J = 2.0 Hz), 7.82 (t, 1H, J = 8.8 Hz), 7.65 (d, 1H,
J = 6.0 Hz), 7.62 (d, 1H, J = 8.8 Hz), 7.21 (dd, 1H, J = 8.8, 2,0 Hz),
4.43 (q, 2H, J = 7.2 Hz), 1.29 (t, 3H, J = 7.2 Hz). ESI-MS: m/z = 397
[M+1]+. Anal. Calcd for C19H16N4O4S: C, 57.57; H, 4.07; N, 14.13.
Found: C, 57.46; H, 4.34; N, 14.22.
The nitro compound 6r (159 mg) was dissolved in EtOH (5 mL)
and Raney Ni (74 mg) was added. The reaction mixture was stirred
at room temperature under H2 for 2 h. Then, the mixture was fil-
tered over Celite, and the filtrate was evaporated to dryness. The
residue was purified by silica gel column chromatography (PE/
EtOAc/EtOH, 3:3:1, v/v/v), yielded pure compound 3-amino-N-(5-
6.1.1.13. 2-Methoxy-N-(5H-c-carboline-8-yl)naphthalene-1-sul-
fonamide (6m). White solid (68%), mp: 205–207 °C. 1H NMR
(d, DMSO-d6): 11.60 (br s, 1H, NH), 9.20 (s, 1H), 8.36 (d, 1H,
J = 5.6 Hz), 8.27 (d, 1H, J = 1.6 Hz), 7.97 (d, 1H, J = 9.2 Hz), 7.91
(m, 2H), 7.72 (dd, 1H, J = 8.8, 2.0 Hz), 7.38 (m, 2H), 7.32 (d, 1H,
J = 8.4 Hz), 7.22 (dd, 1H, J = 8.8, 2.0 Hz), 7.12 (dd, 1H, J = 8.4,
1.6 Hz), 3.86 (s, 3H). ESI-MS: m/z = 404 [M+1]+. Anal. Calcd for
C22H17N3O3S: C, 65.49; H, 4.25; N, 10.42. Found: C, 65.36; H,
4.45; N, 10.32.
6.1.1.14. 4-Bromo-N-(5-(4-bromophenylsulfonyl)-5H-c-carbo-
line-8-yl)benzenesulfonamide (6n). White solid (87%), mp:
>250 °C. 1H NMR (d, DMSO-d6): 10.60 (br s, 1H, NH), 9.38 (s, 1H),
8.68 (d, 1H, J = 6.0 Hz), 8.14 (d, 1H, J = 6.0 Hz), 8.13 (d, 1H,
J = 8.8 Hz), 7.95 (d, 1H, J = 1.6 Hz), 7.87 (d, 2H, J = 8.8 Hz), 7.75
(m, 4H), 7.69 (d, 2H, J = 8.8 Hz), 7.30 (dd, 1H, J = 8.8, 1.6 Hz). ESI-
MS: m/z = 620 [M+1]+. Anal. Calcd for C23H15Br2N3O4S2: C, 44.46;
H, 2.43; N, 6.76. Found: C, 44.36; H, 2.63; N, 6.72.
ethyl-5H-c-carboline-8-yl)benzenesulfonamide (6x) as a white so-
lid (72%), mp: 238–240 °C. 1H NMR (d, DMSO-d6): 9.21 (s, 1H), 8.44
(d, 1H, J = 5.6 Hz), 7.88 (s, 1H), 7.57 (d, 1H, J = 5.6 Hz), 7.53 (d, 1H,
J = 8.8 Hz), 7.20 (dd, 1H, J = 1.6, 8.8 Hz), 7.08 (t, 1H, J = 8.8 Hz), 6.95
(s, 1H), 6,86 (d, 1H, J = 8.0 Hz), 6.64 (d, 1H, J = 8.0 Hz), 5.44 (br s,
2H), 4.38 (q, 2H, J = 7.6 Hz), 1.29 (t, 3H, J = 7.6 Hz). ESI-MS: m/z =
367 [M+1]+. Anal. Calcd for C19H18N4O2S: C, 62.28; H, 4.95; N,
15.29. Found: C, 62.35; H, 4.86; N, 14.97.
6.1.1.15. 4-Methyl-N-(5-(4-methylphenylsulfonyl)-5H-c-carbo-
line-8-yl)benzenesulfonamide (6o). White solid (90%), mp:
>250 °C. 1H NMR (d, DMSO-d6): 10.33 (br s, 1H, NH), 9.58 (s, 1H),
8.82 (d, 1H, J = 6.4 Hz), 8.68 (d, 1H, J = 6.4 Hz), 8.17 (d, 1H,
J = 8.8 Hz), 8.01 (d, 1H, J = 2.0 Hz), 7.83 (d, 2H, J = 8.0 Hz), 7.73
(d, 2H, J = 8.0 Hz), 7.61 (dd, 1H, J = 2.0, 8.8 Hz), 7.30 (d, 2H,
J = 8.0 Hz), 7.23 (d, 2H, J = 8.0 Hz), 2.37 (s, 3H), 2.35 (s, 3H). ESI-
MS: m/z = 492 [M+1]+. Anal. Calcd for C25H21N3O4S2: C, 61.08; H,
4.31; N, 8.55. Found: C, 61.36; H, 4.15; N, 8.52.
6.1.1.19.
2,5-Dimethoxy-N-(5-ethyl-5H-c-carboline-8-yl)ben-
zenesulfonamide (6s). White solid (66%), mp: 177–179 °C. 1H
NMR (d, DMSO-d6): 10.20 (br s, 1H, NH), 9.82 (s, 1H), 8.69 (d, 1H,
J = 6.4 Hz), 8.22 (s, 1H), 8.19 (d, 1H, J = 6.4 Hz), 8.02 (m, 2H), 7.83
(d, 1H, J = 8.4 Hz), 7.46 (d, 1H, J = 8.0), 6.86 (s, 1H), 4.56 (q, 2H,
J = 6.4 Hz), 3.84 (s, 3H), 3.66 (s, 3H), 1.34 (t, 3H, J = 6.4 Hz).
ESI-MS: m/z = 412 [M+1]+. Anal. Calcd for C21H21N3O4S: C, 61.30;
H, 5.14; N, 10.21. Found: C, 61.54; H, 5.47; N, 10.12.
6.1.1.16. 4-Bromo-N-(5-ethyl-5H-c-carboline-8-yl)benzenesul-
fonamide (6p). White solid (56%), mp: 154–156 °C. 1H NMR
(d, DMSO-d6): 9.24 (s, 1H), 8.57 (d, 1H, J = 6.4 Hz), 8.04 (s, 1H),
7.86 (d, 1H, J = 6.4 Hz), 7.62 (d, 2H, J = 8.8 Hz), 7.54 (d, 2H,
J = 8.8 Hz), 7.35 (m, 2H), 4.36 (q, 2H, J = 7.2 Hz), 1.47 (t, 3H, J =
7.2 Hz). ESI-MS: m/z = 430 [M+1]+. Anal. Calcd for C19H16BrN3O2S:
C, 53.03; H, 3.75; N, 9.76. Found: C, 53.24; H, 3.64; N, 9.54.
To a solution of 6p (215 mg, 0.5 mmol) in dry THF–DMF (1 mL,
1:1) was added NaH (18 mg, 0.75 mmol) and the reaction mixture
was stirred for 30 min at ambient temperature. Then C2H5Br
6.1.1.20.
2,4-Dimethoxy-N-(5-ethyl-5H-c-carboline-8-yl)ben-
zenesulfonamide (6t). White solid (79%), mp: 77–79 °C. 1H
NMR (d, DMSO-d6): 9.69 (s, 1H, NH), 9.24 (s, 1H), 8.45 (d, 1H,
J = 6.0 Hz), 7.90 (s, 1H), 7.61 (m, 2H), 7.55 (d, 1H, J = 8.8 Hz), 7.26
(dd, 1H, J = 1.2, 8.8 Hz), 6.64 (d, 1H, J = 2.0 Hz), 6.50 (dd, 1H,
J = 2.0, 8.8 Hz), 4.37 (q, 2H, J = 6.8 Hz), 3.94 (s, 3H), 3.74 (s, 3H),
1.28 (t, 3H, J = 6.8 Hz). ESI-MS: m/z = 412 [M+1]+. Anal. Calcd for
C21H21N3O4S: C, 61.30; H, 5.14; N, 10.21. Found: C, 61.35; H,
(45 lL, 0.6 mmol) was added into the reaction mixture and stirred
4.97; N, 10.44.
for 30 min. Then water was added into the residue and extracted
with EtOAc (3 ꢁ 10 mL). The organic phase was washed with brine
(2 ꢁ 10 mL), dried over anhydrous Na2SO4 and concentrated under
vacuum. The product was purified by silica column chromatogra-
phy using PE/EtOAc/EtOH (5:5:1, v/v/v) as eluent to afford 4-bro-
6.1.1.21.
3,4-Dimethoxy-N-(5-ethyl-5H-c-carboline-8-yl)ben-
zenesulfonamide (6u). White solid (87%), mp: 68–70 °C. 1H
NMR (d, DMSO-d6): 9.97 (s, 1H, NH), 9.27 (s, 1H), 8.46 (d, 1H,
J = 6.0 Hz), 7.97 (d, 1H, J = 2.0 Hz), 7.60 (m, 2H), 7.28 (m, 2H),
7.22 (dd, 1H, J = 2.0, 8.8 Hz), 7.01 (d, 1H, J = 8.4 Hz), 4.39 (q, 2H,
J = 6.8 Hz), 3.74 (s, 3H), 3.69 (s, 3H), 1.29 (t, 3H, J = 6.8 Hz).
ESI-MS: m/z = 412 [M+1]+. Anal. Calcd for C21H21N3O4S: C, 61.30;
H, 5.14; N, 10.21. Found: C, 61.59; H, 4.96; N, 10.24.
mo-N-ethyl-N-(5-ethyl-5H-c-carboline-8-yl)benzenesulfonamide
(6y) as a white solid (31%), mp: 103–105 °C. 1H NMR (d, DMSO-d6):
9.33 (s, 1H), 8.50 (d, 1H, J = 5.6 Hz), 8.04 (d, 1H, J = 1.6 Hz), 7.82
(d, 2H, J = 8.4 Hz), 7.69 (d, 1H, J = 8.8 Hz), 7.66 (d, 1H, J = 5.6 Hz),
7.54 (d, 2H, J = 8.4 Hz), 7.15 (dd, 1H, J = 8.8, 1.6 Hz), 4.48 (q, 2H,

8484
J. Chen et al. / Bioorg. Med. Chem. 18 (2010) 8478–8484
6.1.1.22. 2,4,6-Trimethoxy-N-(5-ethyl-5H-
c-carboline-8-yl)ben-
bated with purified bovine tubulin and buffer containing 20% glyc-
zenesulfonamide (6v). White solid (68%), mp: 223–224 °C. 1H
NMR (d, DMSO-d6): 9.56 (s, 1H, NH), 9.18 (s, 1H), 8.45 (d, 1H,
J = 5.6 Hz), 7.93 (s, 1H), 7.58 (m, 2H), 7.30 (dd, 1H, J = 2.0, 8.8 Hz),
6.19 (s, 2H), 4.38 (q, 2H, J = 6.8 Hz), 3.84 (s, 6H), 3.74 (s, 3H), 1.29
(t, 3H, J = 6.8 Hz). ESI-MS: m/z = 442 [M+1]+. Anal. Calcd for
erol and 1 mM GTP at 37 °C and the effect of compounds 6p, 6t–v,
6y and CA-4 on tubulin polymerization were monitored kinetically
using a fluorescent plate reader.
6.5. DNA intercalation assay
C22H23N3O5S: C, 59.85; H, 5.25; N, 9.52. Found: C, 59.83; H, 5.22;
N, 9.45.
Purified Lambda DNA marker (Takara) (5
5 mg/mL) were treated with compounds 6p, 6t and 6y (1.25
l
L) and EB (1.25
l
l
L,
L,
6.1.1.23. 4-Phenoxy-N-(5-ethyl-5H-
c
-carboline-8-yl)benzene-
0.2 mg/mL) for 15 min, and then loaded on agarose gel. After elec-
trophoresis, DNA-EB complex was photographed by Bio-Rad
GD2000.
sulfonamide (6w). White solid (68%), mp: 100–102 °C. 1H NMR
(d, DMSO-d6): 10.10 (br s, 1H, NH), 9.27 (s, 1H), 8.47 (d, 1H,
J = 4.8 Hz), 7.93 (s, 1H), 7.72 (d, 2H, J = 8.0 Hz), 7.60 (m, 2H), 7.40
(m, 2H), 7.23 (d, 2H, J = 8.0 Hz), 7.06 (m, 4H), 4.40 (q, 2H,
J = 7.2 Hz), 1.30 (t, 3H, J = 7.2 Hz). ESI-MS: m/z = 444 [M+1]+. Anal.
Calcd for C25H21N3O3S: C, 67.70; H, 4.77; N, 9.47. Found: C,
67.57; H, 4.46; N, 9.74.
6.6. Molecular docking study
Tubulin-DAMAcolchicine crystal structure (PDB ID: 1SA0) was
chosen as a template. The functional A, B chains were kept, polar
hydrogens were added, and CHARMm force field was employed.
Binding sphere (45.4407, 41.1246, 34.6219, 9) was selected from
the active site using the binding site tools. For all tested com-
pounds, hydrogens were added and CHARMm force fields were
employed. Each of the compounds was minimized by Dreiding
Minimize tool. CDOCKER (Discovery Studio 2.1) was used for the
docking simulation. For each compound, the docking parameters
were as follows: Top Hits: 25; Random Conformations: 10; Ran-
dom Conformations Dynamics Steps: 1000; Grid Extension: 8.0;
Random Dynamics Time Step: 0.002. Final docked conformations
were scored by calculated cdocker-energy.
6.1.1.24. 4-Methyl-N-(5-tert-butoxycarbonyl-5H-c-carboline-8-
yl)benzenesulfonamide (6z). White solid (82%), mp: 112–114 °C.
1H NMR (d, DMSO-d6): 9.32 (s, 1H), 8.61 (d, 1H, J = 5.6 Hz), 8.07 (d,
1H, J = 9.2 Hz), 8.05 (d, 1H, J = 5.6 Hz), 7.93 (d, 1H, J = 2.0 Hz), 7.68
(d, 2H, J = 8.0 Hz), 7.30 (d, 2H, J = 8.0 Hz), 7.28 (dd, 1H, J = 9.2,
2.0 Hz), 2.28 (s, 3H), 1.67 (s, 9H). ESI-MS: m/z = 438 [M+1]+. Anal.
Calcd for C23H23N3O4S: C, 63.14; H, 5.30; N, 9.60. Found: C,
63.33; H, 5.24; N, 9.36.
6.2. Cytotoxic assay
The tumor cell lines (A549, SGC, HCT116, MCF-7 and K562)
were obtained from Shanghai Institute of Pharmaceutical Industry.
The cytotoxic activity in vitro was measured using the MTT as-
Acknowledgments
This study was financially supported by the National Key Tech
Project for Major Creation of New drugs (No. 2009ZX09501-003)
and the Fundamental Research Funds for the Central Universities
(No. KYJD038).
say.17 MTT solution (10.0
lL/well) in RPMI-1640 (Sigma, St. Louis,
MO) was added after cells were treated with drug for 48 h, and
cells were incubated for a further 4 h at 37 °C. The purple formazan
crystals were dissolved in 100.0 lL DMSO. After 5 min, the plates
were read on an automated microplate spectrophotometer (Bio-
Tek Instruments, Winooski, VT) at 570 nm. Assays were performed
in triplicate in three independent experiments. The concentration
required for 50% inhibition of cell viability (IC50) was calculated
using the software ‘Dose-Effect Analysis with Microcomputers’.
The tumor cell line panel consisted of A549, SGC, HCT116, MCF-
7, and K562. In all of these experiments, three replicate wells were
used to determine each point.
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Bioorg. Med. Chem. 2009, 17, 3324.
For flow cytometry analysis of DNA content, A549 cells in expo-
nential growth were treated with 6p (2
washed twice with PBS and fixed in 75% ethanol. The cell pellet
was resuspended in 100.0 L of PBS containing 200.0 mg/mL RNase
lM) for 48 h. Cells were
l
(Amersco, Solon, OH), then incubated at 37 °C for 0.5 h. After incu-
bation, the cells were stained with 20.0 mL/L propidium iodide (PI,
Sigma, St. Louis, MO) for 15 min. The fluorescence cell was mea-
sured with FACSCalibur (Becton–Dickinson, Lincoln Park, NJ).
14. Cao, R.; Peng, W.; Wang, Z.; Xu, A. Curr. Med. Chem. 2007, 14, 479.
15. Chen, J.; Chen, W. L.; Hu, Y. Synlett 2008, 1, 77.
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Knossow, M. Nature 2004, 428, 198.
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6.4. Microtubule polymerization assay19
In vitro tubulin polymerization assays were conducted with re-
agents as described by the manufacturer (Cytoskeleton, Inc). In
brief, compounds 6p, 6t–v, 6y (4 lM) and CA-4 (3 lM) were incu-