Furanylazaindoles: Potent anticancer agents in vitro and in vivo

Article abstract of DOI:10.1021/jm4011115

Preliminary biological data on 7-anilino-6-azaindoles (8-11) suggested that hydrophobic substituents at C7 contribute to enhancement of antiproliferative activity. A novel series of 7-aryl-6-azaindole-1-benzenesulfonamides (12-22) were developed and showe

Full text of DOI:10.1021/jm4011115

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Article
Furanyl-azaindoles: Potent Anticancer Agents in vitro and in vivo.
Hsueh-Yun Lee, Shiow-Lin Pan, Min-Chieh Su, Yi-Min Liu, Ching-Chuan
Kuo, Yi-Ting Chang, Jian-Sung Wu, Chih-Ying Nien, Samir Mehndiratta, Chi-
Yen Chang, Su-Ying Wu, Mei-Jung Lai, Jang-Yang Chang, and Jing-Ping Liou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4011115 • Publication Date (Web): 24 Sep 2013
Downloaded from http://pubs.acs.org on October 9, 2013
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Page 1 of 34
Journal of Medicinal Chemistry
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N
N
5
6
O S
2
O
OMe
7
21, IC = 32 nM (HT29)
50
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Hydrophobic
interaction
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Revised Manuscript for Article
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Furanyl-azaindoles: Potent Anticancer Agents in vitro and in vivo.
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Hsueh-Yun Lee,† Shiow-Lin Pan,¶ Min-Chieh Su,† Yi-Min Liu,† Ching-Chuan Kuo,‡,║ Yi-Ting Chang,†
Jian-Sung Wu,║ Chih-Ying Nien,† Samir Mehndiratta,† Chi-Yen Chang, ‡ Su-Ying Wu,║ Mei-Jung Lai†
Jang-Yang Chang,*, ₸ Jing-Ping Liou*,†
‡,
School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei 11031,
Taiwan, Republic of China. National Institute of Cancer Research, National Health Research Institutes,
Tainan, Taiwan, Republic of China. The Ph.D program for Cancer Biology and Drug Discovery,
College of Medical Science and Technology, Taipei Medical University. Taipei, Taiwan, Republic of
China. Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes,
Miaoli, Taiwan, Republic of China. Division of Hematology/Oncology, Department of Internal
Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung
University, Tainan, Taiwan, Republic of China.
* To whom correspondence should be addressed. For J. Y. Chang: phone, 886-6-700-0123 ext. 65100; E-mail,
jychang@nhri.org.tw. For J. P. Liou: phone, 886-2-2736-1661 ext 6130; E-mail, jpl@tmu.edu.tw.
†School of Pharmacy, College of Pharmacy, Taipei Medical University.
‡National Institute of Cancer Research, National Health Research Institutes.
¶ The Ph.D program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical
University.
║Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes.
₸
Division of Hematology/Oncology, Department of Internal Medicine, National Cheng Kung University Hospital, College of
Medicine, National Cheng Kung University
Abbreviations: VDA, vascular disrupting agent; CA-4, combretastatin A-4; MDR, multidrug resistant; MRP, multidrug
resistant-associated protein; Pd(PPh3)4, Tetrakis(triphenylphosphine)palladium(0).
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Journal of Medicinal Chemistry
ABSTRACT
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Preliminary biological data on 7-anilino-6-azaindoles (8–11) suggested that hydrophobic substituents at
C-7 contribute to enhancement of antiproliferative activity. A novel series of 7-aryl-6-azaindole-1-
benzenesulfonamides (12–22) were developed and showed improved cytotoxicity compared to ABT751
(5). The conversion of C-7 phenyl rings into C-7 heterocycles led to a remarkable improvement of
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antiproliferative
activity.
Among
all
the
synthetic
products,
7-(2-Furanyl)-1-(4-
methoxybenzenesulfonyl)-6-azaindole (21) exhibited the most potent anticancer activity against KB,
HT29, MKN45, and H460 cancer cell lines with IC50 values of 21.1, 32.0, 27.5, and 40.0 nM,
respectively. Bioassays indicated that 21 not only inhibits tubulin polymerization by binding to tubulin
at the colchicine binding site but also arrests the cell cycle at the G2/M phase with slight arrest at the
sub-G1 phase. Compound 21 also functions as a vascular disrupting agent and dose-dependently inhibits
tumor growth without significant change of body weight in an HT29 xenograft mouse model. Taken
together, compound 21 has potential for further development as a novel class of anticancer agents.
INTRODUCTION
One of the four phases of cell cycle, the M-phase, in which two daughter cells separate and which is
involved with the formation of mitotic spindles is one of the most common targets for the development
of anticancer agents. The mitotic spindle component consists of microtubules which act as guides that
direct chromosomes into individual cells and is therefore a crucial target for development of antimitotic
agents. In addition to the role of microtubules in cell proliferation, their other significant functions are
the subject of intensive investigation. Several potent antimitotic agents are known including colchicine
(1), combretastatin A4 (CA-4, 2), and their prodrugs like CA-4P (3) and ombrabulin (4),1 as well as
numerous synthetic molecules.2 In view of the diverse structural characteristics of antimitotic agents,3
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researchers are frequently attracted to their study and to the challenge of using them as lead compounds
in the development of new potent antiproliferative agents.
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6
7
H CO
H CO
3
3
NHCOCH
O
3
8
9
H CO
3
H CO
3
CH O
OCH
3
3
R
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OCH
3
OCH
3
1: Colchicine
2: Combretastatin A-4 (CA-4), R = OH
3: Combretastatin A-4P (CA-4P), R = OPO Na
4: Ombrabulin, R = NH-serine
3
2
-
O
+
N
NHSO
NH
OCH
3
2
Cl
COCH
N
NH
3
N
O
2
S
SO
2
N
H
H CO
3
SO NH
2
2
OH
6: HMN-214
7: Indisulam
5: ABT751
Figure 1. Natural antimitotic agents and their derivatives and synthetic antitumor agents.
ABT751 (5) was identified in 1992 as a potent antiproliferative agent from its ability to arrest the cancer
cell cycle.4 Advanced investigations of 5 showed that it inhibits cancer cell growth by arresting the
G2/M phase of the cell cycle. Indisulam (E7070, 7), derived by modification of 5, was identified as a
potent anticancer agent which impinges on the cell cycle at both the G1/S and the G2/M phases.6 HMN-
214 (6), a prodrug which is converted in vivo to the unsubstituted sulfonamide, lacking the N-acetyl
group, inhibits cancer cell growth by interfering with the action of the polo-like kinase 1 (PLK1).7 These
synthetic molecules, all sulfonamides, are currently undergoing clinical trials.
7-Aroylaminoindoline-1-benzenesulfonamides8 and 7-arylindoline-1-benzenesulfonamides9 are
recognized as highly potent inhibitors of tubulin polymerization, and serve to identify the [6,5]-bicyclic
heterocycle as the central core of new synthetic antitubulin agents. The structural comparison of the
potent antiproliferative agents cited above indicates that the 4-methoxybenzenesulfonamido motif is
crucial and it is therefore the basis of the compounds in the current study.
Compounds 8–11 were synthesized and their antiproliferative activity was examined. Although the 6-
azaindole derived from ring-closure of 5 was slightly less potent, 6-azaindole was assumed to be a
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Journal of Medicinal Chemistry
suitable structure for further development because examination of various C-7 substituents revealed that
larger hydrophobic groups contributed to an improved potency, triggering our interest in the exploration
of the effect of substitution at the C7 position (Figure 2). A series of 7-aryl- and 7-heteroaryl-6-
azaindole-1-benzenesulfonamides (12–22) were synthesized and the relevant biological assays are
discussed in this paper.
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N
NH
SO
2
NH
HO
OCH
3
Bioisosterism
restricted approach
Ring-closing causes
a slight lost of potency
N
N
O S
2
NH
8: R = H
9: R = OH
10: R = OCH
11: R = F
R
3
OCH
3
6-Azaindole favors C7
hydrophobic moiety
after ring-closing
Removal of polar amino group
N
N
N
N
O S
X
2
O S
2
R
OCH
3
OCH
3
20: X = 4'-pyridinyl
21: X = 2'-furanyl
22: X = 2'-thiophenyl
12: R = H
16: R = N(CH )
3 2
13: R = OH
14: R = OCH
15: R = F
17: R = Cl
18: R = CF
3
3
19: R = NO
2
Figure 2. Rational design of compounds 8–22.
RESULTS AND DISCUSSION
Chemistry. The synthetic route to 7-anilino-6-azaindole-1-benznesulfonamides 8–11 is shown in
Scheme 1. The starting material, 2-bromo-3-nitropyridine (23) was treated with vinylmagnesium
bromide under conditions of the Bartoli indole synthesis yielding 7-bromo-6-azaindole (24).10 Reaction
of 24 with various anilines in the presence of pyridine gave 7-anilino-6-azaindoles (25–27) and was
followed by reaction with 4-methoxybenzenesulfonyl chloride, yielding compounds 8, 10, and 11.
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Compound 24 underwent substitution at N-1 when treated with 4-methoxybenzenesulfonyl chloride and
this was followed by the nucleophilic attack by 4-hydroxyaniline, affording 9. The synthetic routes to
the 7-aryl-6-azaindole-1-benzenesulfonamides (12–22) are shown in Scheme 2. Treatment of 28 with
various phenylboronic acids under conditions of the Suzuki reaction yielded the designed compounds
12–22.11
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Scheme 1. Synthetic Approaches to 7-anilino-6-azaindole-1-benzenesulfonamides 8–11a
N
N
N
a
b
N
c
H
N
N
N
O S
NH
NO
2
2
NH
H
OCH
Br
Br
24
3
R
R
23
25: R = H
8: R = H
26: R = OCH
27: R = F
10: R = OCH
11: R = F
3
3
N
N
c
N
b
N
O S
2
O S
2
NH
Br
OCH
OCH
3
3
HO
28
9
aReagents and conditions: (a) vinylmagnesium bromide, THF, -40 ‒ -50 oC, 60%; (b) anilines, pyridine, 120–130 oC, 35–
52%; (c) 4-methoxybenzenesulfonyl chloride, Bu4NHSO4, KOH, CH2Cl2, room temperature, 18–79%.
Scheme 2. Synthetic Approaches to 7-Aryl-6-azaindole-1-benzenesulfonamides 12–22 a
a
N
N
N
N
O S
2
O S
2
R
Br
OCH
OCH
3
3
12: R = phenyl
28
13: R = 4-hydroxyphenyl
14: R = 4-methoxyphenyl
15: R = 4-fluorophenyl
16: R = 4-N,N-dimethylphenyl
17: R = 4-chlorophenyl
18: R = 4-trifluoromethylphenyl
19: R = 4-nitrophenyl
20: R = 4-pyridinyl
21: R = 2-furanyl
22: R = 2-thiophenyl
aReagents and conditions: (a) substituted phenylboronic acid, Pd(PPh3)4, K2CO3, toluene/EtOH, reflux, 20–63%.
Biological Evaluation.
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A. In Vitro Cell Growth Inhibitory Activity.
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9
In an attempt to evaluate the effect of 7-anilino- and 7-aryl-6-azaindole-1-benzenesulfonamides on
cancer cell growth inhibition, we evaluated all synthesized compounds (8–22) together with the
reference compounds, colchicine and 5, for their antiproliferative activity against four human cancer cell
lines, cervical carcinoma KB cells, colorectal carcinoma HT29 cells, stomach carcinoma MKN45 cells,
and lung carcinoma H460 cells. The results are shown in Table 1.
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Table 1. IC50 Values of Compounds 8–22 and Colchicine
N
N
O S
2
R
OCH
3
IC50 ± SDa (nM)
HT29
compd
R
KB
MKN45
H460
anilino
672.0 ± 193.7 764.5 ± 60.1
607.0 ± 226.3
705.5 ± 94.0
716.0 ± 226.3
671.7± 155.2
297.5 ± 143.5
264.0 ± 169.7
367.5 ± 24.7
410.5 ± 3.5
8
4-OH-anilino
4-MeO-anilino
4-F-anilino
phenyl
649.0 ± 33.9 2771.0 ± 2221.7 913.5 ± 108.2
9
602.3 ± 136.3 535.0 ± 140.3
231.5 ± 65.8 282.0 ± 117.4
245.0 ± 140.0 223.0 ± 117.4
351.5 ± 13.4 314.5 ± 70.0
408.0 ± 50.9 559.5 ± 198.7
358.0 ± 50.0
199.5 ± 33.2
155.5 ± 96.9
253.0 ± 53.7
279.5 ± 20.5
59.5 ± 13.4
10
11
12
13
14
15
16
4-OH-phenyl
4-MeO-phenyl
4-F-phenyl
95.5 ± 16.3
83.0 ± 2.8
91.0 ± 15.6
4-Me2N-phenyl 967.0 ± 32.5 1071.5 ± 181.7
1550.0 ± 353.6 1018.5 ± 115.3
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4-Cl-phenyl
4- CF3-phenyl
4-NO2-phenyl
4-pyridinyl
2-furanyl
282.0 ± 9.9
303.5 ± 7.8
213.0 ± 18.4
186.5 ± 4.9
94.5 ± 11.2
98.5 ± 16.3
27.5 ± 3.5
363.0 ± 17.0
453.9 ± 47.0
222.5 ± 10.0
192.5 ± 20.5
40.0 ± 14.1
120.5 ± 2.1
17
1
2
3
4
5
6
7
8
390.2 ± 20.9 247.0 ± 18.4
172.0 ± 49.5 82.0 ± 8.9
184.0 ± 36.8 138.0 ± 24.2
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19
9
20
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21.1 ± 11.2
72.3 ± 57.6
32.0 ± 7.1
21
2-thiophenyl
–
106.0 ± 5.7
97.5 ± 3.5
22
colchicine
10.3 ± 0.9
15.2 ± 0.5
11.5 ± 1.5
166 ± 8.5
19.8 ± 0.1
–
251.3 ± 64.9 338.7 ± 118.5
217.7 ± 3.8
5
aSD: standard deviation, all experiments were independently performed at least three times.
The comparison of compounds 8–11 with 5 permits assessment of whether 6-azaindole could be a
suitable central core. Compound 9 bearing the same 4-hydroxyanilino group as 5 exhibited 3- to 9-fold
less antiproliferative activity than 5 against a panel of cell lines The conversion of 4-OH to 4-H (8) and
4-OMe (10) gave bioassay results similar to those obtained from 9. Compound 11, with a 4-
fluoroanilino group, tested against four cancer cell lines, exhibited potency comparable to that of 5 with
IC50 values ranging from 199 to 297 nM. Although ring-closure led to a slight loss of activity, this
could be counteracted by substitution at C7 by a hydrophobic motif such as 4-fluorophenyl. This
phenomenon encouraged us to remove the polar amino linkage, and a series of 7-aryl- and 7-heteroaryl-
6-azaindole-1-benzenesulfonamides (12–22) were synthesized. The comparison of 8, 10 and 11 with
compounds 12–15 revealed that the removal of the amino linkage produced an approximately 3-fold
improvement of antiproliferative activity. Compound 15 with a 4-fluorophenyl substituent, exhibits
remarkable activity against KB, HT29, MKN45, and H460 cells with IC50 values of 95, 83, 59, and 91
nM, respectively, and is therefore about 3 times more potent than 5. The bioassay data from compounds
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16–19 suggested a significant effect of diverse substitution influencing inhibition of cancer cell growth.
The introduction of 4-N,N-dimethylaminophenyl (16) for example, led to a dramatic loss of
cytotoxicity, and replacement of fluoro with chloro (17) or trifluoromethyl (18) led to 3- to 4-fold
decrease of antiproliferative potency respectively. Compound 19, with a strong electron-withdrawing
group (NO2) exhibited marked anticancer activity against the four cancer cell lines that were examined,
especially HT29 and MKN45 cells, with IC50 values ranging from 82 to 223 nM. Three heterocyclic
rings (pyridine, furan, and thiophene) were selected based on the isosterism concept and evaluated for
antiproliferative activity. Compound 20, possessing a pyridine ring, has a 2-fold decrease of
cytotoxicity compared to 15. Compound 22 exhibits antiproliferative activity against KB, HT29,
MKN45, and H460 cancer cell lines with IC50 values of 72.3, 106.0, 97.5, and 120.5 nM, respectively.
Thus, replacement of pyridine by thiophene (22) leads to similar cytotoxicity against MKN45 and H460
cells to that of 20, and 22 is slightly more potent against KB and HT29 cells. The replacement of
thiophene with furan resulted in 21 which shows a remarkable improvement of cellular activity, and is
the most potent of all the synthetic molecules. Compound 21 exhibits potent anticancer activity against
KB, HT29, MKN45, and H460 cancer cell lines with IC50 values of 21.1, 32.0, 27.5, and 40.0 nM,
respectively but was slightly less potent than CA-4 or the colchicines. The anticancer cytotoxicity of 21
is much more potent than that of 5.
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A major mechanism of acquired drug resistance is the overexpression of the efflux pumps P-
gp170/MDR and MRP. The efficacy of compounds 15 and 21 against P-gp170/MDR and MRP-
overexpressing drug-resistant cell lines is shown in Table 2. Unlike the classical microtubule inhibitors
colchicine, paclitaxel, and vincristine and the topoisomerase II inhibitor VP-16, 15 and 21 were
equipotent toward the parental KB cells and those of KB-derived MDR-positive cell lines, even in the
presence of a high-level expression of drug-resistant efflux protein (MDR/P-gp or MRP) in KB-Vin 10,
KB-S15, and KB-7D cells. The IC50 values of compounds 15 and 21 for a variety of KB-derived MDR-
positive cells range from 20 to 80 nM. The results also indicated that resistant cells were more sensitive
to compounds 15 and 21 than to 5.
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1
2
3
Table 2. Growth Inhibition of Compounds 15 and 21 against KB-derived MDR-positive lines with different resistance
4
5
phenotypes
6
7
8
9
KB
KB-VIN10
KB-S15
KB-7D
MRP (+)
51.8  8.1
1.2  0.4
7.9  0.5
54  3.5
Growth inhibition
constant (IC50)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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27
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(parental)
10.4  2.5
0.6  0.2
4.1  1.6
1.1  0.2
252  42
78.0  4.1
40.7  8.7
P-gp170/MDR (+) P-gp170/MDR (+)
Colchicine (nM)
Vincristine (nM)b
Paclitaxel (nM)b
VP-16 (M)b
5 (nM)
122  9.4
90.1  7.4
16500  707
23  3
35.4  3.8
17.6  0.5
273  15
3.5  0.3
206  29
85.4  7.2
24.6  1.7
227  27
205  15
66.9  5.6
20.3  2.6
15 (nM)
60.1  1.9
29.2  1.2
21 (nM)
aCells were treated with various concentrations of the test compounds at for three generation times. Cell survival was
determined by methylene blue assay and the IC50 value was calculated. Each value represents the mean  S.D. of three
independent experiments. bData from ref. 12.
B. Inhibition of Tubulin Polymerization and Colchicine Binding.
To investigate whether the activities of the synthetic products in the current study were related to the
interactions with microtubule systems, compounds 12, 16–22, and reference compounds colchicine, 5,
and CA-4 were evaluated as tubulin polymerization inhibitors and for [3H]-colchicine binding activity
and the results are presented in Table 3. Compound 19 showed the best tubulin inhibitory activity with
IC50 value of 1.7 µM, and is thus more potent than CA-4. However, the [3H]colchicine-binding assay
indicated that 19 has only a weak binding affinity for the colchicine binding site. Compounds 21 and 22
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exhibited tubulin inhibitory activity comparable to that of CA-4, but displayed weaker colchicine
binding affinity than CA-4 (Table 3). Compound 21 is however much more potent than 5 in inhibition
of tubulin assembly in vitro. In addition, we found that compound 21 competes with [3H]colchincine in
binding to tubulin. The binding capacity of compound 21 to the colchicine binding site of tubulin is
stronger than that of colchicine or 5, but less than that of CA-4. The Ki values for colchicine, CA-4, 5,
and compound 21 are 2.4, 0.12, 1.51, and 0.38 μM, respectively.
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Table 3. Inhibition of Tubulin Polymerization and Colchicine Binding Inhibition by Compounds 12, 16–22
Inhibition of [3H] colchicine bindingb
Tubulina
Colchicine binding (%)
Compound
IC50 (M)
Ki
1 M
42
5 M
63
3.4
2.4
2.5
2.6
1.7
2.9
2.0
1.9
4.2
2.1
3.3
-
12
41
74
-
16
41
72
-
-
17
35
65
18
21
46
-
19
35
59
-
20
63
81
0.38
-
21
22
30
70
Colchicine
CA-4
5
32
72
2.4
0.12
1.51
87
95
41
73
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aInhibition of tubulin polymerization.13 bInhibition of [3H] colchicine binding.14 Concentration of Tubulin was 1 µM;
Concentration of[3H]colchicine was 5 µM.
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Figure 3. Effect of compounds 15, 19, 21 and 22 on in vitro tubulin polymerization. MAP-rich tubulins were incubated at 37
°C with either a control (dimethyl sulfoxide, DMSO) or test compounds (colchicine or serial concentrations of compounds
15, 19, 21 and 22). The absorbance at 350 nm was measured every 30 s for 30 min and is presented as increases in the
polymerized microtubule.
C. Computational Study.
The molecular docking study was performed to elucidate the interactions of this class of compounds
with tubulin. Compound 21 was docked into the colchicine binding site of tubulin by GOLD 5.1 using
the structure of tubulin in complex with 5 as the template. The docking study (Figure 4) showed that the
methoxybenzene group of compound 21 superimposed with that of 5 and the azaindole group of 21
occupied a similar position as the pyridine group of 5. The azaindole group of 21 made the close
contacts with the surrounding residues, including Cys241, Leu248, Leu255, Ala316, Ala317, Val318
and Ile378. Moreover, the furanyl group ( the substitution at the C7 position) of compound 21 extended
into the hydrophobic cavity of the colchicine binding site formed by Val238, Cys241, Leu242, Ala250,
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Journal of Medicinal Chemistry
Leu252, and Leu255. The furanyl group of compound 21 made strong hydrophobic interactions with
Cys241, Leu242, Ala250, and Leu255 and formed very few polar interactions with the tubulin, which
might explain why the substitution at the C7 position of this class of compounds favors a hydrophobic
moiety.
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Figure 4. Superimposition of the structure of 21 (cyans) and 5 (gray) in the colchicine binding site of tubulin. The furanyl
group of compound 21 occupied hydrophobic cavity and made hydrophobic interactions with Cys241, Leu242, Ala250, and
Leu255.
D. Flow Cytometric Analysis.
The effect of compound 21 on cell cycle progression of KB cells was examined by flow cytometry
(Figure 5). Treatment of compound 21 treatment results in a concentration-dependent accumulation of
KB cells in the G2/M phase with concomitant losses from the G1 and S-phase. In addition, a
characteristic hypodiploid DNA content peak (sub-G1), indicated as apoptotic cells, was also detected
and was also dose-dependent. The value of sub-G1 phase reaches a peak at 100 nM concentrations of 21.
These results indicate cell cycle arrest in G2/M phase induced by compound 21 and occurrence of
apoptotic cell death.
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Figure 5. The concentration effect of compound 21 on cell cycle progress of KB cells. Cells were treated with different
concentrations of compound 21 for 24 h then analyzed for propidium iodide-stained DNA content by flow cytometry.
E. Fluorescence Images of Cell Morphology.
The effect of compound 21 on cellular microtubule networks was examined with immunofluorescence
techniques (Figure 6). In the absence of drug treatment, the microtubule network exhibits normal
arrangement and organization in KB cells but after 6 h, 1 μM of colchicine causes significant cellular
microtubule depolymerization and gives a diffuse α-tubulin stain (Figure 6B). In contrast, 1 μM of
paclitaxel dramatically promotes microtubule polymerization with an increase in the density of cellular
microtubules and formation of long thick microtubule bundles (Figure 6C) while treatment with
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compound 21 produces results similar to those of colchicine-induced microtubule change. We observed
an almost complete loss of microtubules with only a diffuse stain visible throughout the cytoplasm
(Figure 6D–6F).
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Figure 6. Effect of compound 21 on the organizations of cellular microtubule network. KB cells were treated with test
compound for 6 h. After incubation, cells were harvested and fixed, then reacted with monoclonal anti--tubulin antibody at
room temperature for 2 h. After reacting with FITC-conjugated secondary antibody, the cellular microtubules were observed
by fluorescence microscopy.
F. Investigation of Vascular Disrupting Activity.
Antitubulin agents and synthetic flavonoids are classified as small molecular vascular disrupting
agents (VDA).15 Among antitubulin agents, CA-4P (3) is a well-recognized example of a vascular
disrupting agent which causes vascular shutdown within solid tumors.16 Compound 21, was further
investigated for vascular disrupting activity using the following methods. Human umbilical vein
endothelial cells (HUVECs) were plated on Matrigel and allowed to form capillaries in the presence of
vascular endothelial growth factor (VEGF) at a concentration of 20 ng/mL, followed by exposure to test
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compounds, including 5-fluorouracil (5-FU), cisplatin, 5, and compound 21.17 As shown in Figure 7,
clinically used chemotherapeutic drugs, such as 5-FU and cisplatin, at a dosage of 1000 nM had no
affect on tube formation in HUVECs. Significantly however, compound 21 potently disrupts formation
of capillaries in a concentration-dependent manner while having no affect on cell viability, and this
disruption is greater than that caused by 5 at the same concentration.
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Figure 7. Investigation of the vascular disrupting activity of compound 21. HUVECs were plated on Matrigel and allowed to
form capillary tubes in the presence of VEGF (20 ng/mL) followed by exposure to different concentrations of compound 21.
Cultures were photographed, and the number of capillary tube networks was counted under a microscope (original
magnification of 100×). Data reflect the mean number of capillary tube networks compared to the vehicle control group
(DMSO) ± the standard deviation (SD) from three separate experiments. 5-FU and 5 were used as internal controls to
compare the vascular disrupting activity of compound 21.
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G. Growth Inhibition of Human Colon Cancer Xenografts in vivo.
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8
We evaluated the in vivo efficacy of compound 21 using an HT-29 xenograft nude mice model (Figure
8). Once a tumor was approximately 50 mm3 in size and was palpable, the mice were randomized into
vehicle control and treatment groups (7-8 mice per group). Control mice received the vehicle (1.0%
carboxymethyl cellulose +0.5% Tween80). In this study, we demonstrated that the growth of HT-29
cancer cells xenografts is suppressed by factors of 54.0% and 48.1% (percent tumor growth inhibition
[TGI] values) after oral administration of compound 21 at concentrations of 200 mg/kg and 100 mg/kg,
respectively (Figure 8). This tumor growth inhibition is dose-dependent. No significant differences in
body weight or other adverse effects were observed upon treatment with compound 21 (Figure 8).
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Figure 8. Anticancer activity of compound 21 in a xenograft model of human colorectal HT-29 cancer cells. Left panel,
tumor growth of HT-29 xenografts in nude mice treated with or without compound 21 (200, 100, and 50 mg/kg). Tumor
growth is tracked by the mean tumor volume (mm3) ± S.E. The effects on % tumor growth inhibition determined in this study.
Tumor volume was determined using caliper measurements and was calculated as the product of 1/2 x length x width2. Right
panel, body weight (g) of the mice. *, p < 0.05 and ** p < 0.01 as compared with the control group.
Conclusions
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Our preliminary conclusion, that C-7 hydrophobic groups are favored in the series of 6-azaindole-1-
benzenesulfonamides led us to introduce a variety of aryl/heteroaryl groups in place of the phenyl at the
C7 position of compound 12. The biological activities of all the resulting synthetic 7-aryl- and 7-
heteroaryl-6-azaindole-1-benzenesulfonamides were examined. Among all synthetic compounds 21,
with a furan substituent at the C-7 position, exhibits noteworthy potency against a set of cancer cell lines.
With a mean IC50 value of 30 nM against these cancer cell lines, 21 is 10-fold more potent than 5.
Compound 21 induces cell cycle arrest in the G2/M phase and leads to occurrence of apoptotic cell
death. Furthermore, it behaves as a vascular disrupting agent and exhibits activity superior to that of 5
and treatment with 21 exhibits substantial dose-dependent inhibitory activity against HT-29 cells in
xenograft mice. In summary, these newly developed compounds with a novel 7-aryl-6-azaindole
skeleton showed marked biological activity in vitro and in vivo, and have potential for further
development as a novel class of anticancer agents.
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Experimental Section
(A) Chemistry. Nuclear magnetic resonance (1H NMR) spectra were obtained with a Bruker DRX-500
spectrometer operating at 500 MHz, with chemical shift reported in parts per million (ppm, δ) downfield
from TMS, the internal standard. High-resolution mass spectra (HRMS) were measured with a JEOL
(JMS-700) electron impact (EI) mass spectrometer. The purity of the final compounds was determined
with an Agilent 1100 series HPLC system using a C-18 column (Agilent ZORBAX Eclipse XDB-C18 5
μm. 4.6 mm × 150 mm) and was found to be ≥ 95%. Flash column chromatography was done using
silica gel (Merck Kieselgel 60, No. 9385, 230-400 mesh ASTM). All reactions were carried out under
an atmosphere of dry nitrogen.
7-Anilino-1-(4-methoxybenzenesulfonyl)-6-azaindole (8)
KOH (0.08g, 1.43 mmol) and tetra-n-butylammonium hydrogen sulfate (0.02 g, 0.05 mmol) were added
to a solution of 25 (0.10 g, 0.48 mmol) in CH2Cl2 (5 mL) under N2, and the reaction was stirred for 30
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min. 4-Methoxybenzenesulfonyl chloride (0.20 mg, 0.96 mmol) was added slowly to the reaction
mixture. After 1 h the mixture was quenched with H2O then extracted with CH2Cl2. The combined
organic layer was dried over MgSO4, filtered and concentrated. The crude product was purified with
silica gel column chromatography (1:3 EtOAc/n-hexane) to give 31 mg (17％) of 8 as a solid, mp
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138.5−139.7 °C; H NMR (500 MHz, CDCl3) δ 3.75 (s, 3H), 6.62 (d, J = 3.5 Hz, 1H), 6.80 (d, J = 8.9
Hz, 2H), 6.90 (d, J = 5.5 Hz, 1H), 7.05 (t, J = 7.4, 7.5 Hz, 1H), 7.36 (t, J = 7.7, 8.0 Hz, 2H), 7.66 (d, J =
8.9 Hz, 2H), 7.71 (d, J = 3.7 Hz, 1H), 7.74 (d, J = 7.7 Hz, 2H), 7.99 (d, J = 5.3 Hz, 1H), 9.19 (s, 1H);
13C NMR (125 MHz, CDCl3) δ 55.5, 108.0, 109.6, 114.5, 119.0, 119.2, 121.8, 128.4, 128.8, 129.0,
131.2, 139.6, 140.5, 141.1, 143.9, 163.7. MS (EI) m/z 379.1 (M+, 30%), 208.1 (100%); HRMS (EI) for
C20H17N3O3S (M+) calcd. 379.0990, found 379.0990.
7-(4-Hydroxyanilino)-1-(4-methoxybenzenesulfonyl)-6-azaindole (9)
The title compound was obtained in 15% overall yield from compound 28 in a manner similar to that
described for the preparation of 8: 1H NMR (500 MHz, CDCl3) δ 3.78 (s, 3H), 6.62 (d, J =3.7 Hz, 1H),
6.72 (d , J = 8.7 Hz, 2H), 6.84 (d, J = 9.1 Hz, 2H), 6.85 (d, J = 5.4 Hz, 1H), 7.30 (d, J = 8.7 Hz, 2H),
7.68 (d, J = 9.0 Hz, 2H), 7.72 (d, J = 3.7 Hz, 1H), 7.89 (d, J = 5.4 Hz, 1H), 8.85 (s, 1H); MS (EI) m/z
395.2 (M+, 37%) 224.1 (100%); HRMS (EI) for C20H17N3O4S (M+) calcd. 395.0934, found 395.0934.
7-(4-Methoxyanilino)-1-(4-methoxybenzenesulfonyl)-6-azaindole (10)
The title compound was obtained as a solid in 79% overall yield from compound 26 in a manner similar
1
to that described for the preparation of 8, mp 155.5−158.3 °C; H NMR (500 MHz, CDCl3): δ 3.78 (s,
3H), 3.83 (s, 3H), 6.62 (d, J = 3.7 Hz, 1H), 6.83 (d, J = 9.0 Hz, 2H), 6.85 (d, J = 5.4 Hz, 1H), 6.94 (d, J
= 8.9 Hz, 2H), 7.58 (d, J = 8.9 Hz, 2H), 7.68 (d, J = 9.0 Hz, 2H), 7.70 (d, J = 3.6 Hz, 1H), 7.94 (d, J =
5.4 Hz, 1H), 9.03 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 55.5, 55.6, 107.4, 109.7, 114.2, 114.6, 118.8,
121.9, 128.6, 129.2, 131.2, 133.6, 139.6, 141.3, 144.6, 155.2, 163.8; MS (EI) m/z 409.2 (M+, 100%),
238.1 (81%); HRMS (EI) for C21H19N3O4S (M+) calcd. 409.1097, found 409.1098.
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7-(4-Fluoroanilino)-1-(4-methoxybenzenesulfonyl)-6-azaindole (11)
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9
This compound was obtained in a manner similar to that described for the preparation of 8 in 71%
overall yield from compound 27 as a solid, mp 125.1−127.1 °C; 1H NMR (500 MHz, CDCl3) δ 3.74 (s,
3H), 6.62 (d, J = 3.6 Hz, 1H), 6.80 (d, J = 9.0 Hz, 2H), 6.89 (d, J = 5.3 Hz, 1H), 7.05 (dd, J = 8.7, 8.7
Hz, 2H), 7.65 (d, J = 9.0 Hz, 2H), 7.69 (dd, J = 9.0, 4.4 Hz, 2H), 7.69 (d, J = 4.0 Hz, 1H), 7.95 (d, J =
5.2 Hz, 1H), 9.14 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 55.6, 108.0, 109.7, 114.6, 115.3, 115.5, 118.9,
121.2, 128.5, 129.1, 131.3, 136.5, 139.7, 141.1, 144.0, 157.0, 159.4, 163.9; MS (EI) m/z 397.1 (M+,
58%), 226.1 (100%); HRMS (EI) for C20H16N3O3SF (M+) calcd. 397.0895, found 397.0895.
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7-Phenyl-1-(4-methoxybenzenesulfonyl)-6-azaindole (12)
A mixture of 28 (0.10 g, 0.27 mmol) in toluene (8 mL), tetrakis(triphenylphosphine) palladium (0.02 g,
0.02 mmole), 2 M of K2CO3 (1 mL), phenylboronic acid (0.04 g, 0.3 mmol), and EtOH (5 mL) was
heated to reflux under N2 for 24 h. The solvent was then removed under reduced pressure and the
resulting residue was purified with flash chromatography (2:3 EtOAc/n-hexane) to give the 12 (26.6 mg,
1
27 %) as a solid, mp 148.9−151.0 °C; H NMR (500 MHz, CDCl3) δ 3.79 (s, 3H), 6.73 (d, J = 8.9 Hz,
2H), 6.76 (d, J = 3.7 Hz, 1H), 7.18 (d, J = 8.9 Hz, 2H), 7.37 (t, J = 7.1, 7.5 Hz, 3H), 7.42 (d, J = 5.1 Hz,
1H), 7.46 (d, J = 6.9 Hz, 2H), 7.88 (d, J = 3.7 Hz, 1H), 8.44 (d, J = 5.3 Hz, 1H); MS (EI) m/z 364.1 (M+,
36%), 193.1 (100%); HRMS (EI) for C20H16N2O3S (M+) calcd. 364.0872, found 364.0873.
7-(4-Hydroxyphenyl)-1-(4-methoxybenzenesulfonyl)-6-azaindole (13)
Compound 13 was obtained in 35% overall yield from compound 28 in a manner similar to that
1
described for the preparation of 12 as a solid, mp 179−182 °C; H NMR (500 MHz, CDCl3) δ 3.79 (s,
3H), 6.70 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 9.3 Hz, 2H), 6.74 (d, J = 3.0 Hz, 1H), 7.19 (d, J = 8.9 Hz, 2H),
7.27 (d, J = 9.7 Hz, 2H), 7.38 (d, J =5.1 Hz, 1H), 7.88 (d, J = 3.7 Hz, 1H), 8.41 (d, J = 5.1 Hz, 1H); MS
(EI) m/z 380.1 (M+, 33%), 209.1 (100%); HRMS (EI) for C20H16N2O4S (M+) calcd. 380.0831, found
380.0831.
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1-(4-Methoxybenzenesulfonyl)-7-(4-methoxyphenyl)-6-azaindole (14)
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The title compound was obtained in 84% overall yield from compound 28 in a manner similar to that
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7
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described for the preparation of 12 as a solid, mp 162.8−164.8 °C; H NMR (500 MHz, CDCl3) δ 3.79
8
9
(s, 3H), 3.88 (s, 3H), 6.73 (d, J = 3.8 Hz, 1H), 6.73 (d, J = 8.9 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 7.21 (d,
J = 9.0 Hz, 2H), 7.34 (d, J = 5.2 Hz, 1H), 7.43 (d, J = 8.7 Hz, 2H), 7.84 (d, J = 3.7 Hz, 1H), 8.41 (d, J =
5.1 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 55.1, 55.5, 109.0, 112.9, 113.8, 114.0, 128.7, 128.8, 130.5,
130.9, 132.6, 134.0, 139.7, 142.6, 148.1, 159.6, 163.4; MS (EI) m/z 394.1 (M+, 25%), 223.1 (100%);
HRMS (EI) for C21H18N2O4S (M+) calcd. 394.0990, found 394.0991.
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7-(4-Fluorophenyl)-1-(4-methoxybenzenesulfonyl)-6-azaindole (15)
Compound 15 was obtained in 81% overall yield from compound 28 in a manner similar to that
1
described for the preparation of 12 as a solid, mp 136.0−137.0 °C; H NMR (500 MHz, CDCl3) δ 3.80
(s, 3H), 6.75 (d, J = 8.8 Hz, 2H), 6.75 (d, J = 4.2 Hz, 1H), 7.03 (t, J = 8.6, 8.7 Hz, 2H), 7.20 (d, J = 8.9
Hz, 2H), 7.40 (d, J = 5.1 Hz, 1H), 7.43 (ddd, J = 8.3, 8.3, 2.7 Hz, 2H), 7.85 (d, J = 3.7 Hz, 1H), 8.42 (d,
13
J = 5.1 Hz, 1H); C NMR (125 MHz, CDCl3) δ 55.6, 108.9, 114.0, 114.4, 114.6, 128.8, 131.0, 134.1,
136.2, 139.8, 142.6, 147.2, 161.6, 163.6, 164.0; MS (EI) m/z 382.1 (M+, 63%), 211.1 (93%); HRMS (EI)
for C20H15N2O3SF (M+) calcd. 382.0783, found 382.0783.
7-(4-(N,N-dimethylaminophenyl))-1-(4-methoxybenzenesulfonyl)-6-azaindole (16)
The title compound was obtained in 61% overall yield from compound 28 in a manner similar to that
1
described for the preparation of 12 as a solid, mp 111.5−114.5 °C; H NMR (500 MHz, CDCl3) δ 3.02
(s, 6H), 3.77 (s, 3H), 6.70 (d, J = 9.2 Hz, 2H), 6.71 (d, J = 8.7 Hz, 2H), 6.72 (d, J = 3.8 Hz, 1H), 7.24 (d,
J = 8.8 Hz, 2H), 7.25 (d, J = 5.1 Hz, 1H), 7.45 (d, J = 8.7 Hz, 2H), 7.80 (d, J = 3.6 Hz, 1H), 8.38 (d, J =
5.1 Hz, 1H); MS (EI) m/z 407.2 (M+, 20%), 236.1 (100%); HRMS (EI) for C22H21N3O3S (M+) calcd.
407.1305, found 407.1305.
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7-(4-Chlorophenyl)-1-(4-methoxybenzenesulfonyl)-6-azaindole (17)
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The title compound was obtained in 13% overall yield from compound 28 in a manner similar to that
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described for the preparation of 12 as a solid, mp 125.5−128.5 °C; H NMR (500 MHz, CDCl3) δ 3.81
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(s, 3H), 6.76 (d, J = 3.8 Hz, 1H), 6.76 (d, J = 8.8 Hz, 2H), 7.20 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 8.5 Hz,
2H), 7.35 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 5.3 Hz, 1H), 7.86 (d, J = 3.8 Hz, 1H), 8.42 (d, J = 5.1 Hz, 1H);
13C NMR (125 MHz, CDCl3) δ 55.6, 108.8, 114.0, 114.9, 127.7, 128.7, 128.9, 130.6, 130.7, 134.1,
134.3, 138.4, 139.8, 142.5, 146.9, 163.6; MS (EI) m/z 398.1 (M+, 56%), 227.0 (42%), 171.0 (100%);
HRMS (EI) for C20H15N2O3SCl (M+) calcd. 398.0499, found 398.0499.
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1-(4-Methoxybenzenesulfonyl)-7-(4-trifluoromethylphenyl)-6-azaindole (18)
The title compound was obtained in 42% overall yield from compound 28 in a manner similar to that
described for the preparation of 12 as a solid, mp 139.1-140.0 °C; 1H NMR (500 MHz, CDCl3) δ 3.82
(s, 3H), 6.76 (d, J = 8.91 Hz, 2H), 7.79 (d, J = 3.68 Hz, 1H), 7.20 (d, J = 8.69 Hz, 2H), 7.47 (d, J = 5.11
Hz, 1H), 7.64 (d, J = 8.67 Hz, 2H), 7.83 (d, J =3.71 Hz, 1H), 8.21 (d, J = 8.62 Hz, 2H), 8.48 (d, J = 5.08
Hz, 1H); MS (EI) m/z 409.0 (M+, 32.0%), 171.0 (100.0%); HRMS (EI) for C20H15N3O5S (M+) calcd.
409.0732, found 409.0730.
1-(4-Methoxybenzenesulfonyl)-7-(4-nitrophenyl)-6-azaindole (19)
The title compound was obtained in 31% overall yield from compound 28 in a manner similar to that
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described for the preparation of 12 as a solid, mp 177.5−178.8 °C; H NMR (500 MHz, CDCl3) δ 3.82
(s, 3H), 6.76 (d, J = 8.91 Hz, 2H), 7.79 (d, J = 3.68 Hz, 1H), 7.20 (d, J = 8.69 Hz, 2H), 7.47 (d, J = 5.11
Hz, 1H), 7.64 (d, J = 8.67 Hz, 2H), 7.83 (d, J =3.71 Hz, 1H), 8.21 (d, J = 8.62 Hz, 2H), 8.48 (d, J = 5.08
Hz, 1H); MS (EI) m/z 409.0 (M+, 32.0%), 171.0 (100.0%); HRMS (EI) for C20H15N3O5S (M+) calcd.
409.0732, found 409.0730.
1-(4-Methoxybenzenesulfonyl)-7-(pyridin-4-yl)-6-azaindole (20)
The title compound was obtained in 41% overall yield from compound 28 in a manner similar to that
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described for the preparation of 12 as a solid, mp 110.5−113.2 °C; H NMR (500 MHz, CDCl3) δ 3.81
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(s, 3H), 6.72−6.79 (m, 3H), 7.19 (d, J = 8.93 Hz, 2H), 7.32 (dd, J = 4.49 Hz, 2H), 7.87 (d, J = 3.62 Hz,
1H), 8.46 (d, J = 5.12 Hz, 1H), 8.60 (d, J = 5.93 Hz, 2H); MS (EI) m/z 365.0 (M+, 100.0%); HRMS (EI)
for C19H15N3O3S (M+) calcd. 365.08341, found 365.0836.
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7-(2-Furanyl)-1-(4-methoxybenzenesulfonyl)-6-azaindole (21)
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The title compound was obtained in 72% overall yield from compound 28 in a manner similar to that
described for the preparation of 12 as a solid, mp 136.3-137.8 °C; 1H NMR (500 MHz, CDCl3) δ 3.79 (s,
3H), 6.56 (m, 1H), 6.70 (d, J = 3.70 Hz, 1H), 6.80 (d, J = 8.94 Hz, 2H), 6.83 (d, J = 3.31 Hz, 1H), 7.36
(d, J = 5.14 Hz, 1H), 7.42 (d, J = 9.02 Hz, 2H), 7.74 (d, J = 3.71 Hz, 1H), 8.43 (d, J = 5.08 Hz, 1H); MS
(ESI) m/z 377.0 (M+Na); HRMS (ESI) for C18H15N2O4S (M + H+): calcd. 355.0753; found, 355.0763.
1-(4-Methoxybenzenesulfonyl)-7-(2-thiophenyl)-6-azaindole (22)
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The title compound was obtained as a solid in 65% overall yield from compound 28 in a manner similar
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to that described for the preparation of 12, mp 110.1-110.3 °C; H NMR (500 MHz, CDCl3) δ 3.78 (s,
3H), 6.70 (d, J = 3.72 Hz, 1H), 6.75 (d, J = 8.89 Hz, 2H), 7.05 (t, J = 4.34 Hz, 1H), 7.28 (d, J = 8.88 Hz,
2H), 7.31 (d, J = 5.07 Hz, 1H), 7.34 (d, J = 3.88 Hz, 1H), 7.43 (d, J = 5.00 Hz, 1H), 7.81 (d, J = 3.66 Hz,
1H), 8.39 (d, J = 5.07 Hz, 1H); MS (EI) m/z 393.0 (M+Na+, 36.0%); MS (ESI) m/z 393.0 (M+Na);
HRMS (ESI) for C18H15N2O3S2 (M + H+): calcd. 371.0524, found, 371.0503.
7-Bromo-6-azaindole (24)
Vinylmagnesium bromide (1.0 M in THF, 40 mL, 40 mmole) was added to a mixture of 2-bromo-3-
nitropyridine (2.00 g, 9.7 mmole) in THF (80 mL) at -78 oC and the mixture was stirred at -40 oC − -50
oC for an additional 1 h. The reaction was quenched with saturated NaHCO3 then extracted with EtOAc
(3 times), and the combined organic layer was dried over MgSO4, filtered and concentrated. The
resulting residue was purified by silica gel column chromatography (1:3 EtOAc/n-hexane) to afford 24
(1100 mg, 60％); 1H NMR (500 MHz, CDCl3) δ 6.66 (dd, J = 2.9, 2.1 Hz, 1H), 7.43 (dd, J = 2.9, 2.1 Hz,
1H), 7.51 (d, J = 5.2 Hz, 1H), 8.03 (d, J = 5.3 Hz, 1H), 8.79 (br, 1H)
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7-Anilino-6-azaindole (25)
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A solution of 24 (0.2 g, 1.02 mmol), aniline (0.38 g, 4.06 mmol) in pyridine (1 mL) was heated in a
sealed round flask at 120-130 °C for 24 h. The solvent was removed under reduced pressure and the
resulting residue was purified by flash chromatography (1:2 EtOAc/n-hexane) to give 25 (110 mg, 52
%); 1H NMR (500 MHz, CDCl3) δ 6.51 (d, J = 3.0 Hz, 1H), 7.05 (t, J = 7.2, 7.5 Hz, 1H), 7.12 (d, J = 7.4
Hz, 2H), 7.18 (d, J = 3.1 Hz, 1H), 7.21 (d, J = 5.6 Hz, 1H), 7.30 (dd, J = 7.5, 7.6 Hz, 2H), 7.91 (d, J =
5.6 Hz, 1H).
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7-(4-Methoxyanilino)-6-azaindole (26)
The title compound was obtained in 41% overall yield from the reaction of compound 24 with 4-
1
methoxyaniline in a manner similar to that described for the preparation of 25: H NMR (500 MHz,
CDCl3) δ 3.71 (s, 3H), 6.39(d, J = 3.1 Hz, 1H), 6.79(d, J = 9.0 Hz, 2H), 6.98(d, J = 5.7 Hz, 1H), 7.15(d,
J = 3.1 Hz, 1H), 7.30(d, J = 8.8 Hz, 2H), 7.63(d, J = 5.8 Hz, 1H).
7-(4-Fluoroanilino)-6-azaindole (27)
The title compound was obtained in 35% overall yield from the reaction of compound 24 with 4-
1
fluoroaniline in a manner similar to that described for the preparation of 25: H NMR (500 MHz,
CDCl3): δ 6.50 (d, J = 3.1 Hz, 1H), 6.88 (dd, J = 8.6, 8.7 Hz, 2H), 7.05 (dd, J = 8.9, 4.7 Hz, 2H), 7.15 (d,
J =2.8 Hz, 1H), 7.16 (d, J = 5.6 Hz, 1H), 7.83 (d, J = 5.7 Hz, 1H).
7-Bromo-1-(4-methoxysulfonyl)-6-azaindole (28)
The title compound was obtained in 97% overall yield from compound 24 in a manner similar to that
described for the preparation of 8: 1H NMR (500 MHz, CDCl3): δ 3.84 (s, 3H), 6.72 (d, J = 3.7 Hz, 1H),
6.94 (d, J = 8.9 Hz, 2H), 7.46 (d, J = 5.1 Hz, 1H), 7.77 (d, J = 8.9 Hz, 2H), 8.07 (d, J = 3.7 Hz, 1H),
8.12 (d, J = 5.2 Hz, 1H).
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Journal of Medicinal Chemistry
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(B) Biology
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(a) Materials. Regents for cell culture were obtained from Gibco-BRL Life Technologies (Gaitherburg,
MD). Microtubule-associated protein (MAP)-rich tubulin was purchased from Cytoskeleton, Inc.
(Denver, CO). [3H]Colchicine (specific activity, 60-87 Ci/mmol) was purchased from PerkinElmer Life
Sciences (Boston, MA).
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(b) Cell Growth Inhibitory Assay. Human cancer cell lines (KB, MKN45, HT29, and H460) used in
this study were procured from American Type Culture Collection (Rockville, MD) and grown in
Dulbecco’s modified Eagle’s medium, minimal essential medium, or RPMI 1640 medium. Resistant
cell lines KB-Vin10, KB-7D, and KB-S15 were maintained in a medium containing an additional 10
nM vincristine, 7 µM VP16, or 50 nM paclitaxel, respectively. All cell cultures were supplemented with
10% fetal bovine serum, 2 µM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and
incubated in a humidified atmosphere (95% air and 5% CO2) at 37 °C. KB-Vin10 and KB-S15 were cell
lines resistant against vincristine and paclitaxel, respectively, and both overexpressed the MDR drug
efflux protein. KB-7D cells were VP16-resistant cells and overexpressed MRP. All resistant cell lines
were incubated in a drug-free medium for 3 days before harvesting for use in the growth inhibition
assay. In vitro growth inhibition was assessed with the methylene blue assay.18 In brief, exponentially
growing cells were seeded into 24-well culture plates at a density of 8000 to 20,000 cells/ml/well
(depending on the doubling time of the cell line) and allowed to adhere overnight. Cells were incubated
with various concentrations of drugs for 72 h. Then, we measured the absorbance at 595 nm (A595 nm) of
the resulting solution from 1% N-lauroylsarcosine extraction. The 50% growth inhibition (IC50) was
calculated on the basis of the A595 nm of untreated cells (taken as 100%). The values shown are the means
and SEs of at least three independent experiments performed in duplicate.
(c) Tubulin Polymerization in Vitro Assay.19 Turbidimetric assays of microtubules were performed as
described by Bollag et al.20 Briefly, microtubule-associated protein (MAP)-rich tubulin (from bovine
brain, Cytoskeleton, Denver, C.O.) were dissolved in reaction buffer (100 mM PIPES (pH 6.9), 2 mM
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MgCl2, 1 mM GTP) in preparing a 4 mg/mL tubulin solution. Tubulin solution (240 g MAP-rich
tubulin per well) was placed in 96-well microtiter plate in the presence of test compounds or 2% (v/v)
DMSO as a vehicle control. The increase in absorbance was measured at 350 nm in a PowerWave X
Microplate Reader (BIO-TEK Instruments, Winooski, VT) at 37 °C and recorded every 30 s for 30 min.
The area under the curve (AUC) used to determine the concentration that inhibited tubulin
polymerization to 50% (IC50). The AUC of the untreated control and 10 μM of colchicine were set to
100% and 0% polymerization, respectively, and the IC50 was calculated by nonlinear regression in at
least three experiments.
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(d) Computational Study. The crystal structure of Tubulin in complex with ABT-751 (PDB code
3HKC) was used as the template in the docking study. The protein structure was prepared by SYBYL-X
Suite (version 1.2, Tripos) to add and minimize hydrogens. The bound inhibitor and waters were
removed before docking. Docking was performed with GOLD Suite software package (version 5.1,
CCDC, Cambridge, UK) and scored by the GOLD fitness scoring function. The region within a radius
of 12 Å centered on the SG atom of Cys241 was defined as the active site for docking study. Thirty
genetic algorithm (GA) runs were used for 21 docking. The standard default parameter settings such as
population size 100, selection pressure 1.1, number of operations 100,000, number of islands 5, niche
size 2, crossover 95, mutation 95, and migration 10 were adapted for docking process except the early
termination option was set to off. The annealing parameters of van der Waals and H-bond interactions
were applied within 4.0 and 2.5 Å , respectively, to allow poor geometry at the beginning of a GA run.
(e) Tubulin Competition-Binding Scintillation Proximity Assay. The colchicine competition-binding
scintillation proximity assays were conducted as described previously using biotin-labeled tubulin and
streptavidin-labeled poly(vinyl toluene) SPA beads.21-23 This assay was performed in a 96-well plate. In
brief, 0.08 μM of [3H]colchicine was mixed with the test compound and 0.5 μg special long-chain
biotin-labeled tubulin (0.5 μg) and the mixture was incubated in 100 μL of reaction buffer (80 mM
PIPES, pH 6.8, 1 mM EGTA, 10% glycerol, 1 mM MgCl2, and 1 mM GTP) for 2 h at 37 °C. Then
eighty μg of Streptavidin-labeled SPA beads were added to each reaction. The radioactive counts were
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Journal of Medicinal Chemistry
measured directly with a scintillation counter. Inhibition constants (Ki) were calculated using the Cheng-
Prusoff equation.24
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(f) Capillary Disruption assays.17 Capillary disruption assays were carried out in a 96-well plate
format using human umbilical vein endothelial cells (HUVECs) plated at 2×104 cells per well in 20%
FBS M199 medium containing 20 ng/mL VEGF on a Matrigel layer (BD Biosciences). Capillaries were
allowed to form over a 4 h period before the addition of test compound or vehicle control. Images were
acquired immediately following compound addition and 4 h after exposure to test compound. Tube
formation was quantified manually by counting the network number of capillary structures under a
microscope (original magnification 100X).
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(g) Immunofluorescence Microscopy. Cells attached to poly(L-lysine)-coated coverslips were treated
with drugs for 24 h. Cells were fixed in MeOH/Me2CO (1:1 v/v) at 20 °C for 1 h and then washed with
PBST for 5 min. Nonspecific sites were blocked by incubating with 5% skim milk in phosphate-
buffered saline/tween (PBST) for 1 h. A mouse monoclonal antibody against α-tubulin was diluted
1:500 in blocking solution and incubated for 2 h. Cells were washed with PBST twice (10 min each) to
remove excess antibody and then probed with FITC-conjugated secondary antibody (1:200) for 1 h at
room temperature. The images of α-tubulin with FITC staining were captured with an Olympus BX50
fluorescence microscope (Olympus, Dulles, VA).
(h) Analysis of Cell Cycle Distribution. KB cells were initially seeded at 1 × 106 cells in 100 mm2
dishes and then incubated with various concentrations of test compound for 24 h. Cellular DNA was
stained with PBS containing 50 μg/mL propidium iodide (PI) and 50 μg/mL RNase in the dark at room
temperature for 20 min. The cell cycle was determined by a fluorescence-activated cell sorting IV flow
cytometer (BD Biosciences, Franklin Lakes, NJ) with 10,000 cells scored. Data were analyzed and
graphs were prepared using the Modfit 2.0 program (Verity Software House,Topsham, ME).
(i) Immunofluorescence Staining. KB cells plated on coverslips were treated with indicated
concentration of tested compound for 6 h. After treatment, cells were fixed in MeOH/Me2CO (1:1 v/v)
at -20 oC for 1 h and then washed with PBS. Subsequently, the cells were blocked with 5% skim milk in
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PBS for 1 h then further incubated with 5% skim milk containing anti--tubulin monoclonal antibody
(1:500 dilution) for 2 h at room temperature. Cells were washed with PBS twice (10 min each) to
remove excess antibody and then probed with FITC-conjugated secondary antibody (1:200 dilution) for
1 h at room temperature. The images of cellular microtubules were captured with an Olympus BX50
fluorescence microscope (Dulles, VA). For comparison, paclitaxel and colchicines were also included,
along with compound 21.
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(j) Antitumor Activity in vivo. Nude athymic mice (female, 7-8 weeks) were subcutaneously injected
with 1 × 107 HT-29 cells per mouse. When tumor volumes reached approximately 50 mm3, mice were
randomized by tumor size into 4-5 treatment groups as follows: (control group) vehicle (1.0%
carboxymethyl cellulose/0.5% Tween-80 in ddH2O). Treatment groups: 200 mg/kg, 100 mg/kg, and 50
mg/kg compound 21 administered orally once every day. Tumor volumes were calculated using caliper
measurements twice per week using the formula volume (mm3) = (length × width2)/2. Body weights
were measured daily during the first week and then twice per week.
Acknowledgment. This research were supported by the National Science Council of the Republic of
China (grant no. NSC 100-2628-M-038-001-MY3, and NSC 101-2325-B-038 -002).
Supporting Information Available: The HPLC results and 1H-NMR spectrum of target compounds 8-
22. This material is available free of charge via the Internet at http://pubs.acs.org.
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