Synthesis and biological evaluation of 7-arylindoline-1-benzenesulfonamides as a novel class of potent anticancer agents

Jang-Yang Chang; Mei-Jung Lai; Yi-Ting Chang; Hsueh-Yun Lee; Yun-Ching Cheng; Ching-Chuan Kuo; Min-Chieh Su; Chi-Yen Chang; Jing-Ping Liou

doi:10.1039/C0MD00052C

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DOI: 10.1039/C0MD00052C (Concise Article) Med. Chem. Commun., 2010, 1, 152-155

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Synthesis and biological evaluation of 7-arylindoline-1-benzenesulfonamides as a novel class of potent anticancer agents†

Jang-Yang Chang‡ ^bc, Mei-Jung Lai‡ ^a, Yi-Ting Chang ^a, Hsueh-Yun Lee ^a, Yun-Ching Cheng ^b, Ching-Chuan Kuo ^b, Min-Chieh Su ^a, Chi-Yen Chang ^b and Jing-Ping Liou *^a
^aCollege of Pharmacy, Taipei Medical University, 250 Wu-Xin Street, Taipei 110, Taiwan, Republic of China. E-mail: jpl@tmu.edu.tw; Tel: +886-2-27361661 ext. 6130
^bNational Institute of Cancer Research, National Health Research Institutes, Tainan 704, Taiwan, Republic of China
^cDivision of Hematology/Oncology, Department of Internal Medicine, National Cheng Kung University Hospital, Tainan 704, Taiwan, Republic of China

Received 22nd April 2010 , Accepted 28th April 2010

First published on 18th May 2010

Abstract

A series of 7-arylindoline-1-benzenesulfonamides were prepared and evaluated for anticancer activity. 7-(4′-Cyanophenyl)indoline-1-benzenesulfonamide 15 exhibited substantial antiproliferative activity with IC₅₀ values ranging from 17–32 nM against a variety of human cancer cell lines, including MDR resistant line. Compound 15 (IC₅₀ = 1.5 μM) also showed more potent inhibition of tubulin polymerization than 4a (combretastatin A-4, IC₅₀ = 2.0 μM) and displayed strong binding to the colchicine binding site of the tubulin.

Introduction

Microtubules are dynamic structures that play a crucial role in cellular division and are recognized as an important target for anticancer therapy.¹ A number of naturally occurring compounds, such as paclitaxel, vinblastine, combretastatin A-4 (4a), dolastatin 10, epothilone A and colchicine (6), all exhibit their anticancer properties by interfering with the dynamics of tubulin polymerization and depolymerization, resulting in mitotic arrest. Recent reports² have revealed that compounds binding to the colchicine domain can act as vascular-disrupting agents (VDA), rapidly depolymerizing the microtubules of vasculatures to block the blood supply to tumors; for example, combretastatin A-4P (4b, ZYBRESTAT) and AVE8062 (5b, Ombrabulin) are currently in clinical trials.

Small molecules with sulfonamide functionality, for instance, N-pyridinyl sulfonamide (1, ABT-751),³ chloroindolyl sulfonamide (2, Indisulam),⁴ and styrylpyridine N-oxide sulfonamide (3b, HMN-214),⁵ have been reported as potent anticancer agents and are currently undergoing clinical trials for a variety of cancers (Fig. 1).⁶ Compound 1 showed efficacious inhibition of tubulin polymerization and was found to be a potent antimitotic agent. To our knowledge, there have been no reports on the inhibition of tubulin polymerization with 7-arylindoline-1-benzenesulfonamides. Thus, we report the structure–activity relationship of 7-aryl- and 7-heteroaryl-indoline-1-benzenesulfonamides as a novel class of tubulin depolymerization inhibitors (Fig. 2).


	Fig. 1


	Fig. 2

Results and discussion

Chemistry

Indolines 7–23 and indoles 24–25 were synthesized as shown in Scheme 1, by Suzuki coupling⁷ of various arylboronic acids and heteroarylboronic acids at the 7-position of indoline-1-benzenesulfonamide (28) and indole-1-benzenesulfonamide (29), respectively. The key intermediate 28, 7-bromo-1-(4′-methoxybenzenesulfonyl)indoline, was synthesized starting from the 7-bromoindole (26). The sodium cyanoborohydride-mediated reduction of 26 resulted in the 7-bromoindoline (27), which reacted with the 4-methoxybenzenesulfonyl chloride in pyridine to give 28. Compound 28 was reacted with the appropriate arylboronic acid and heteroarylboronic acid in the presence of Pd(PPh₃)₄ and K₂CO₃ in toluene–EtOH to give the desired 7-aryl(heteroaryl)indoline-1-benzenesulfonamides (7–8 and 10–23). The reduction of the 4′-nitro group in compound 14 with Fe/NH₄Cl in isopropanol gave the corresponding amine 9. Indole-sulfonamides 24 and 25 were obtained from an intermediate, 7-bromo-1-(4′-methoxybenzenesulfonyl)indole (29), which was prepared by treatment of 26 with 4-methoxyphenylsulfonyl chloride in the presence of tetrabutylammonium hydrogen sulfate and KOH in CH₂Cl₂. The Suzuki coupling of 29 with 4-cyanophenylboronic acid and 4-fluorophenylboronic acid gave the desired compounds 24 and 25, respectively.


	Scheme 1 Reagents and conditions: (a) NaCNBH₃, CH₃COOH, rt; (b) 4-methoxybenzenesulfonyl chloride, pyridine, reflux; (c) various aryl or heteroaryl boronic acids, Pd(PPh₃)₄, K₂CO₃, toluene–EtOH, reflux; (d) Fe, NH₄Cl, isopropanol; (e) 4-methoxybenzenesulfonyl chloride, Bu₄NHSO₄, KOH, CH₂Cl₂, rt.

Biological evaluation

(A) In vitro cell growth inhibitory activity. The synthesized 7-arylindoline-1-benzenesulfonamides (7–18), 7-heteroarylindoline-1-benzenesulfonamides (19–23), 7-arylindole-1-benzenesulfonamides (24–25), and reference compounds (1 and 6) were evaluated for their antiproliferative activities against five human cancer cell lines: cervical carcinoma KB cells, colorectal carcinoma HT29 cells, non-small cell lung carcinoma H460 cells, and stomach carcinoma MKN45 cells, as well as an MDR-positive cell line (KB-vin 10) that overexpresses p-gp 170/MDR. (Table 1)

Table 1 Antiproliferative IC₅₀ values (nM ± SD^a) of compounds 7–25, and a reference compound (colchicine) against various cell lines

Compound	Cell type (IC₅₀ nM ± SD^a)
Compound	KB	H460	HT29	MKN45	KB-vin10
a SD: standard deviation. All experiments were independently performed at least three times.
7	119.5 ± 85.6	212.5 ± 67.2	96.5 ± 92.6	116.8 ± 125.6	107.2 ± 18.0
8	337.0 ± 230.5	301.5 ± 170.4	277.0 ± 226.9	211.3 ± 130.5	352.4 ± 25.2
9	249.4 ± 68.7	287.5 ± 98.3	300.5 ± 106.8	161.9 ± 70.0	261.5 ± 41.5
10	538.5 ± 188.8	556.0 ± 183.8	467.0 ± 244.2	237.3 ± 130.6	553.0 ± 128.4
11	3685.5 ± 869.0	6336.0 ± 518.7	5100.0 ± 276.2	1078.7 ± 869.1	4672.3 ± 519.0
12	129.3 ± 40.0	113.5 ± 14.8	133.0 ± 15.9	82.6 ± 36.2	123.2 ± 21.0
13	86.0 ± 15.6	95.5 ± 20.5	85.5 ± 19.1	79.0 ± 28.3	94.6 ± 9.2
14	52.5 ± 4.9	53.1 ± 17.0	51.8 ± 8.5	34.0 ± 9.6	51.3 ± 8.2
15	31.1 ± 5.4	29.5 ± 14.8	24.2 ± 7.2	17.2 ± 8.6	29.2 ± 2.1
16	98.5 ± 12.0	166.5 ± 23.3	119.3 ± 58.6	84.7 ± 24.5	92.4 ± 21.7
17	45.0 ± 7.1	46.0 ± 12.7	57.0 ± 28.3	55.5 ± 14.8	46.0 ± 6.2
18	4040.5 ± 84.1	4755.0 ± 770.7	3897.5 ± 664.0	1777.0 ± 200.8	4632.0 ± 256.0
19	96.0 ± 14.1	101.0 ± 8.0	87.5 ± 40.3	69.5 ± 7.6	106.0 ± 18.0
20	95.9 ± 36.6	89.6 ± 36.2	76.3 ± 26.9	68.5 ± 47.3	84.5 ± 8.0
21	143.0 ± 27.3	135.0 ± 56.0	108.5 ± 81.5	145.3 ± 40.0	150.6 ± 19.4
22	214.5 ± 16.3	229.0 ± 7.1	199.5 ± 6.4	165.0 ± 9.9	251.3 ± 38.2
23	82.0 ± 18.4	94.5 ± 21.9	62.5 ± 2.1	42.0 ± 1.4	79.2 ± 21.3
24	152.9 ± 45.7	171.7 ± 35.3	164.0 ± 66.1	142.7 ± 32.1	146.1 ± 32.6
25	203.1 ± 3.0	211.9 ± 12.6	196.2 ± 10.9	170.2 ± 54.5	201.7 ± 16.5
colchicine	10.3 ± 0.9	19.8 ± 0.1	15.2 ± 0.5	11.5 ± 1.5	121.2 ± 9.6

First, we evaluated whether the substitution of the phenyl group at the 7-position of indoline-1-benzenesulfonamide core would result in antiproliferative activity. 1-(4-Methoxybenzenesulfonyl)-7-phenyl-2,3-dihydro-1H-indole (7) demonstrated potent cell growth inhibitory activity with mean IC₅₀ values of 130 nM (KB, H460, HT29, MKN45, and KB-vin10). Next, investigation of electronic effects on the 7-aryl group of the indoline ring showed that electron-donating groups like 4-methoxyphenyl (8), 4-aminophenyl (9), and 4-(N,N-dimethyl)aminophenyl (10) result in a slight decrease in activity as compared to parent compound 7, exhibiting mean IC₅₀ values of 295, 252 and 470 nM, respectively. It was also noteworthy that the more water soluble 7-(4′-hydroxyphenyl)indoline-1-benzenesulfonamide 12, with the para-hydroxyphenyl group, maintained substantial cytotoxicity, presumably due to the weaker electron-donating ability of the hydroxyl group. As many microtubule inhibitors, such as compounds 4 and 6, have the structure of trimethoxyphenyl group, the addition of trimethoxybenzene moiety may affect activity positively. Compound 11, with a 3′,4′,5′-trimethoxyphenyl group, exhibited dramatically diminished activities with IC₅₀ values ranging from 1078–6336 nM. The steric effect of the substitutions in the 7-arylindoline system seems to influence antiproliferative activity. The important role of the inductive effect on the C7-phenyl moiety of indoline-1-benzenesulfonamides was revealed by the stronger cellular growth inhibition by para(4′)-fluoro-, nitro-, and cyano-substituted phenyl compounds 13, 14 and 15, respectively (mean IC₅₀ values of 88, 49, and 26 nM against the panel of the cell lines), as compared to compounds 7, 8 and 9 with phenyl, 4′-methoxyphenyl and 4′-aminophenyl substitutions. Notably, compound 15, had a mean IC₅₀ value of 26 nM in five cancer cell lines. Compounds 16 and 17 with meta (3′)-fluoro and 3′,4′-difluoro, respectively, were also synthesized and evaluated for antiproliferative activity. SAR information indicated that the fluoro group located at the 3′-position (16) and 4′-position (13) in the 7-arylindoline core resulted in similar activities to each other, but interestingly, the difluoro substitution at the 3′ and 4′-position of phenyl group (17) resulted in improvement of the IC₅₀ values as compared with the mono-fluoro-substituted 13 or 16, with around 0.5–1-fold elevation in potency against five cancer cell lines (KB, H460, HT29, MKN45 and KB-vin10). Notably, compound 17 showed a mean IC₅₀ value of 50 nM in five cancer cell lines, including MDR-positive KB-vin10, which was around 2-fold more potent than the 7-phenylindoline-1-benzenesulfonamides (7). Increase of the number of fluoro groups in pentafluoro-substituted compound 18 resulted in a dramatic decrease in activity to the μM range, thus revealing that steric hindrance on the C7-phenyl regimen of the indoline ring affects the cellular growth inhibitory activity. The improved activity of the 7-aryl-substituted indoline-1-benzenesulfonamides inspired us to investigate the effect of the heteroaryl group at the C-7 position. Compound 19 with a 4′-pyridinyl substitution, compound 20 with a 3′-pyridinyl group, and compound 23 with a 2′-furanyl group at the C-7 position of the indoline ring exhibited stronger antiproliferative activity against human cancer cell lines as compared to compound 7. The 6′-fluoro-3′-pyridinyl (21) and 2′-thiophenyl (22) analogs, however, showed decreased cell growth inhibition as compared to the 7-phenyl compound 7, showing mean IC₅₀ values of 136 and 212 nM, respectively.

In an effort to further understand whether the indoline ring of 7-arylindoline-1-benzenesulfonamides plays an important role for activity, indole sulfonamides 24 and 25 were also prepared. Compounds 24 and 25 showed a decreased growth inhibition by around 2- and 5-fold magnitude in five cancer cell lines, respectively, as compared to the corresponding indolines 15 and 13, thus indicating that the indoline core in this system was preferred over indole (24vs.15 and 25vs.13).

(B) Inhibition of tubulin polymerization and colchicine binding activity. The selected 7-arylindoline-1-benzenesulfonamides (13–15, 17, 19, and 23) and reference compounds (CA4 and colchicine) were evaluated for antitubulin activities in order to investigate whether these compounds were exerting their activities by interactions with microtubules. Specifically, their ability to compete for the colchicine binding site was measured (Table 2). Results indicated that 7-arylindoline-sulfonamide compounds 13, 14, 15, 17, 19 and 23 showed substantial inhibition of tubulin polymerization with IC₅₀ values of 2.5, 1.7, 1.5, 2.3, 2.2 and 2.6 μM, respectively. Notably, compounds 14 and 15 exhibited stronger activity against microtubule assembly than CA4 (IC₅₀ = 2.0 μM). In the [³H]colchicine-binding competition assay, all 7-arylindoline-1-sulfonamides (13–15, 17, 19 and 23) were strongly bound to the colchicine binding domain of tubules.

Table 2 Inhibition of tubulin polymerization and colchicine binding by compounds 13–15, 17, 19, 23 and reference compounds (CA4 and colchicine)

Compound	Tubulin^a IC₅₀ ± SD/μM	Colchicine binding^b (% ± SD)
Compound	Tubulin^a IC₅₀ ± SD/μM	1 μM inhibitor	5 μM inhibitor
a Inhibition of tubulin polymerization.⁸ b Inhibition of [³H]colchicine binding.⁹ Tubulin was at 1 μM; [³H]colchicine was at 5 μM.
13	2.5 ± 0.1	59 ± 3	73 ± 2
14	1.7 ± 0.2	56 ± 3	74 ± 1
15	1.5 ± 0.1	81 ± 2	92 ± 1
17	2.3 ± 0.4	52 ± 2	72 ± 3
19	2.2 ± 0.6	54 ± 3	72 ± 2
23	2.6 ± 0.2	43 ± 2	68 ± 1
colchicine	4.1 ± 0.5
CA4	2.0 ± 0.2	86 ± 1	96 ± 1

Conclusion

We have identified the 7-arylindoline-1-benzenesulfonamides as a novel class of potent inhibitors of tubulin polymerization acting through the colchicine-binding domain of tubulin. The lead compounds 14 and 15 showed significant inhibition of tubulin polymerization with IC₅₀ values of 1.7 and 1.5 μM, respectively, which were superior to CA4. They also demonstrated substantial antiproliferative activity, with IC₅₀ values ranging from 17–54 nM in a variety of human cancer cell lines, including one MDR-positive resistant cell line (KB-vin10). The SAR information of the indoline substitution pattern revealed that the phenyl group located at the 7-position of indoline-1-benzenesulfonamides is beneficial for activity. The electron-withdrawing substituent on the 7-aryl group improved activity (13, 14, 15, 17, 19vs.7). A 2–5-fold decrease in potency was observed on replacing the indoline ring with the indole, thus indicating the important role of the indoline moiety of 7-arylindoline-1-sulfonamides (24vs.15 and 25vs.13). In summary, 7-arylindoline-1-benzenesulfonamides (14 and 15) have potential for further investigation as anticancer agents with strong inhibition of tubulin polymerization and antiproliferative activities.

This research was supported by the National Science Council of the Republic of China (grant no. NSC 98-2323-B-038-003, NSC 98-2113-M-038-002-MY2), Department of Health of the Republic of China (grant no. DOH99-TD-C-111-004), and National Health Research Institutes, Taiwan (grant no. CA-097-PP-02).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Spectral data of compounds 7–25 and experimental procedures for synthesis and biological evaluations, and HPLC purity data for compounds 7–25. See DOI: 10.1039/c0md00052c

‡ Contributed equally to this work.