Furanylazaindoles: Potent Anticancer Agents in Vitro and in Vivo

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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, ChiYen 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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N O

Journal of Medicinal Chemistry

N O2S OMe

21, IC50 = 32 nM (HT29)

Hydrophobic interaction

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Revised Manuscript for 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,*,‡,₸ 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, [email protected]. For J. P. Liou: phone, 886-2-2736-1661 ext 6130; E-mail, [email protected]. †

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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ABSTRACT

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-1benzenesulfonamides (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 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.

H3CO

H3CO NHCOCH3

H3CO CH3O

H3CO OCH3 O OCH3

1: Colchicine

NHSO2 N

2: Combretastatin A-4 (CA-4), R = OH 3: Combretastatin A-4P (CA-4P), R = OPO3Na2 4: Ombrabulin, R = NH-serine

N+

OCH3

5: ABT751

OCl

COCH3

NH

OH

R OCH3

N SO2

NH O2 S N H

H3CO

SO2NH2 6: HMN-214

7: Indisulam

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 HMN214 (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 6azaindole derived from ring-closure of 5 was slightly less potent, 6-azaindole was assumed to be a ACS Paragon Plus Environment

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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-6azaindole-1-benzenesulfonamides (12–22) were synthesized and the relevant biological assays are discussed in this paper.

N NH

NH SO2

HO OCH3 Bioisosterism restricted approach Ring-closing causes a slight lost of potency N

N NH O2S 8: R = H 9: R = OH 10: R = OCH3 11: R = F

R 6-Azaindole favors C7 hydrophobic moiety after ring-closing

OCH3

Removal of polar amino group

N

N

N

R

12: R = H 13: R = OH 14: R = OCH3 15: R = F

N X

O2S

O2S

OCH3

OCH3

16: R = N(CH3)2 17: R = Cl 18: R = CF3 19: R = NO2

20: X = 4'-pyridinyl 21: X = 2'-furanyl 22: X = 2'-thiophenyl

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. ACS Paragon Plus Environment

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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

Scheme 1. Synthetic Approaches to 7-anilino-6-azaindole-1-benzenesulfonamides 8–11a

a

N

NO2 Br

N

b

N

N H

N H

N

c

NH

N NH O2S OCH3

Br R

24

23

R 25: R = H 26: R = OCH3 27: R = F

c

N

N

b

N Br O2S

N NH O2S

OCH3

OCH3 HO

28

a

8: R = H 10: R = OCH3 11: R = F

9

Reagents 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

N

a N Br O2S

28

a

N R

OCH3

N O2S OCH3

12: R = phenyl 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

Reagents and conditions: (a) substituted phenylboronic acid, Pd(PPh3)4, K2CO3, toluene/EtOH, reflux, 20–63%.

Biological Evaluation. ACS Paragon Plus Environment

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A. In Vitro Cell Growth Inhibitory Activity. 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.

Table 1. IC50 Values of Compounds 8–22 and Colchicine

N R

N O2S OCH3

IC50 ± SDa (nM) compd

R

KB

HT29

MKN45

H460

8

anilino

672.0 ± 193.7 764.5 ± 60.1

607.0 ± 226.3

705.5 ± 94.0

9

4-OH-anilino

649.0 ± 33.9

913.5 ± 108.2

716.0 ± 226.3

10

4-MeO-anilino

602.3 ± 136.3 535.0 ± 140.3

358.0 ± 50.0

671.7± 155.2

11

4-F-anilino

231.5 ± 65.8

282.0 ± 117.4

199.5 ± 33.2

297.5 ± 143.5

12

phenyl

245.0 ± 140.0 223.0 ± 117.4

155.5 ± 96.9

264.0 ± 169.7

13

4-OH-phenyl

351.5 ± 13.4

314.5 ± 70.0

253.0 ± 53.7

367.5 ± 24.7

14

4-MeO-phenyl

408.0 ± 50.9

559.5 ± 198.7

279.5 ± 20.5

410.5 ± 3.5

15

4-F-phenyl

95.5 ± 16.3

83.0 ± 2.8

59.5 ± 13.4

91.0 ± 15.6

16

4-Me2N-phenyl

967.0 ± 32.5

1071.5 ± 181.7

1550.0 ± 353.6

1018.5 ± 115.3

2771.0 ± 2221.7


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17

4-Cl-phenyl

282.0 ± 9.9

303.5 ± 7.8

213.0 ± 18.4

363.0 ± 17.0

18

4- CF3-phenyl

390.2 ± 20.9

247.0 ± 18.4

186.5 ± 4.9

453.9 ± 47.0

19

4-NO2-phenyl

172.0 ± 49.5

82.0 ± 8.9

94.5 ± 11.2

222.5 ± 10.0

20

4-pyridinyl

184.0 ± 36.8

138.0 ± 24.2

98.5 ± 16.3

192.5 ± 20.5

21

2-furanyl

21.1 ± 11.2

32.0 ± 7.1

27.5 ± 3.5

40.0 ± 14.1

22

2-thiophenyl

72.3 ± 57.6

106.0 ± 5.7

97.5 ± 3.5

120.5 ± 2.1

colchicine

–

10.3 ± 0.9

15.2 ± 0.5

11.5 ± 1.5

19.8 ± 0.1

5

–

251.3 ± 64.9

338.7 ± 118.5

166 ± 8.5

217.7 ± 3.8

a

SD: 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 4fluoroanilino 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-heteroaryl6-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 ACS Paragon Plus Environment

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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. A major mechanism of acquired drug resistance is the overexpression of the efflux pumps Pgp170/MDR and MRP. The efficacy of compounds 15 and 21 against P-gp170/MDR and MRPoverexpressing 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 MDRpositive 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. ACS Paragon Plus Environment

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Table 2. Growth Inhibition of Compounds 15 and 21 against KB-derived MDR-positive lines with different resistance phenotypes

Growth inhibition

KB

KB-VIN10

KB-S15

constant (IC50)

(parental)

Colchicine (nM)

10.4  2.5

122  9.4

35.4  3.8

51.8  8.1

Vincristine (nM)b

0.6  0.2

90.1  7.4

17.6  0.5

1.2  0.4

Paclitaxel (nM)b

4.1  1.6

16500  707

273  15

7.9  0.5

VP-16 (M)b

1.1  0.2

23  3

3.5  0.3

54  3.5

5 (nM)

252  42

227  27

206  29

205  15

15 (nM)

78.0  4.1

60.1  1.9

85.4  7.2

66.9  5.6

21 (nM)

40.7  8.7

29.2  1.2

24.6  1.7

20.3  2.6

P-gp170/MDR (+) P-gp170/MDR (+)

KB-7D MRP (+)

a

Cells 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 ACS Paragon Plus Environment

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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.

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

5 M

12

3.4

42

63

-

16

2.4

41

74

-

17

2.5

41

72

-

18

2.6

35

65

-

19

1.7

21

46

-

20

2.9

35

59

-

21

2.0

63

81

0.38

22

1.9

30

70

-

Colchicine

4.2

32

72

2.4

CA-4

2.1

87

95

0.12

5

3.3

41

73

1.51


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Inhibition of tubulin polymerization.13 bInhibition of [3H] colchicine binding.14 Concentration of Tubulin was 1 µM;

Concentration of[3H]colchicine was 5 µM.

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, ACS Paragon Plus Environment

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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.

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


13

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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).

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 ACS Paragon Plus Environment

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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.

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. 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).

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 width 2. 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-1benzenesulfonamides 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 7heteroaryl-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.

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 ACS Paragon Plus Environment

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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 138.5−139.7 °C; 1H 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); 13

C 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 to that described for the preparation of 8, mp 155.5−158.3 °C; 1H 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. ACS Paragon Plus Environment

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7-(4-Fluoroanilino)-1-(4-methoxybenzenesulfonyl)-6-azaindole (11) 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. 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, 27 %) as a solid, mp 148.9−151.0 °C; 1H 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 described for the preparation of 12 as a solid, mp 179−182 °C; 1H 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. ACS Paragon Plus Environment

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1-(4-Methoxybenzenesulfonyl)-7-(4-methoxyphenyl)-6-azaindole (14) The title compound was obtained in 84% overall yield from compound 28 in a manner similar to that described for the preparation of 12 as a solid, mp 162.8−164.8 °C; 1H NMR (500 MHz, CDCl3) δ 3.79 (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. 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 described for the preparation of 12 as a solid, mp 136.0−137.0 °C; 1H 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, J = 5.1 Hz, 1H); 13C 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 described for the preparation of 12 as a solid, mp 111.5−114.5 °C; 1H 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) The title compound was obtained in 13% overall yield from compound 28 in a manner similar to that described for the preparation of 12 as a solid, mp 125.5−128.5 °C; 1H NMR (500 MHz, CDCl3) δ 3.81 (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); 13

C 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. 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 described for the preparation of 12 as a solid, mp 177.5−178.8 °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-(pyridin-4-yl)-6-azaindole (20) The title compound was obtained in 41% overall yield from compound 28 in a manner similar to that described for the preparation of 12 as a solid, mp 110.5−113.2 °C; 1H NMR (500 MHz, CDCl3) δ 3.81 ACS Paragon Plus Environment

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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. 7-(2-Furanyl)-1-(4-methoxybenzenesulfonyl)-6-azaindole (21) 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) The title compound was obtained as a solid in 65% overall yield from compound 28 in a manner similar to that described for the preparation of 12, mp 110.1-110.3 °C; 1H 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-3nitropyridine (2.00 g, 9.7 mmole) in THF (80 mL) at -78 oC and the mixture was stirred at -40 oC − -50 o

C 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) 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). 7-(4-Methoxyanilino)-6-azaindole (26) The title compound was obtained in 41% overall yield from the reaction of compound 24 with 4methoxyaniline in a manner similar to that described for the preparation of 25: 1H 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 4fluoroaniline in a manner similar to that described for the preparation of 25: 1H 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). ACS Paragon Plus Environment

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(B) Biology (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). (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 ACS Paragon Plus Environment

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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. (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 ACS Paragon Plus Environment

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measured directly with a scintillation counter. Inhibition constants (Ki) were calculated using the ChengPrusoff equation.24 (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). (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 phosphatebuffered 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 ACS Paragon Plus Environment

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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. (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 822. This material is available free of charge via the Internet at http://pubs.acs.org.


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Table of Contents graphic.

N O

N O2S OMe

21, IC50 = 32 nM (HT29)

Hydrophobic interaction


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Furanylazaindoles: Potent Anticancer Agents in Vitro and in Vivo

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