Concise syntheses of N-aryl-5,6,7-trimethoxyindoles as antimitotic and vascular disrupting agents: Application of the copper-mediated Ullmann-type arylation

Article abstract of DOI:10.1039/c0ob01038c

In an attempt to mimic the 3,4,5-trimethoxyphenyl-Z-stilbene moiety of combretastatin A-4, a series of N-aryl-5,6,7-trimethoxyindoles were synthesized via copper-catalyzed Ullmann-type N-arylation through the corresponding 5,6,7-trimethoxyindole and aryl halides. These synthesized compounds demonstrated potent antiproliferative activity providing a novel skeleton for potent tubulin polymerization inhibitors.

Full text of DOI:10.1039/c0ob01038c

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Concise syntheses of N-aryl-5,6,7-trimethoxyindoles as antimitotic and
vascular disrupting agents: application of the copper-mediated Ullmann-type
arylation†
Hsueh-Yun Lee,‡a Jang-Yang Chang,‡b,c Ling-Yin Chang,a Wen-Yang Lai,b Mei-Jung Lai,a
Kuang-Hsing Shih,b Ching-Chuan Kuo,b Chi-Yen Changb and Jing-Ping Liou*a
Received 17th November 2010, Accepted 24th February 2011
DOI: 10.1039/c0ob01038c
In an attempt to mimic the 3,4,5-trimethoxyphenyl-Z-stilbene
moiety of combretastatin A-4, a series of N-aryl-5,6,7-
trimethoxyindoles were synthesized via copper-catalyzed
Ullmann-type N-arylation through the corresponding 5,6,7-
trimethoxyindole and aryl halides. These synthesized com-
pounds demonstrated potent antiproliferative activity pro-
viding a novel skeleton for potent tubulin polymerization
inhibitors.
Halting the mitotic process of cancer cells through targeting micro-
tubules has been a promising strategy to develop potent anticancer
agents.1 Natural products like colchicine (1) and combretastain
A-4 (CA4, 2) (Fig. 1) have been recognized as antimitotic agents
interfering with the dynamics of tubulin polymerization. These
compounds are known to interact with the colchicine binding site
of microtubules resulting in mitotic arrest. Combretastain A-4 was
discovered in 1988 and was further examined as a potent antimi-
totic agent.2 Due to the simple structure and excellent potency of
Fig. 1
CA4, the distinguishable Z-stilbene and 3,4,5-trimethoxybenzene
motif (A-ring) of CA4 attracted scientists’ efforts to search for
more potent anticancer agents.3 In an attempt to increase the
solubility, for instance, the phosphate prodrug CA4P (3) was
synthesized and is currently in human clinical trails.1,4 Compound
4 which has 3¢-OH of CA4 replaced with 3¢-NH2 demonstrated
potent antitubulin activity5 and its serine prodrug (AVE8602, 5) is
undergoing human clinical trials as well.1a
as a surrogate of the A-ring of CA4. Based on CA4 structure and
our experience, 3¢-hydroxy-4¢-methoxyphenyl, N-methylindolyl6
and quinolinyl7 groups were selected and attached to the N-
1 position of 5,6,7-trimethoxyindole in this study. Therefore, a
series of N-aryl-5,6,7-trimethoxyindoles were synthesized (6–10)
and investigated for antiproliferative activity.
Organometallic chemistry has been comprehensively applied to
construct various heterocycles which serve as central structures
in the field of medicinal chemistry.8 In the lead optimization,
organometallic chemistry played a significant role to carry out
the functionalization as well.9 N-Arylation of indoles from the
corresponding aryl halides and indoles could be carried out
through two approaches, transition metal-catalyzed coupling re-
actions and aromatic nucleophilic substitution reactions (SNAr).10
Palladium and copper are commonly exploited in transition metal-
catalyzed coupling reactions. Compared to palladium-catalyzed
reactions, however, copper provides milder conditions, lower cost
reagents and broader applications.11 To achieve our designed
N-arylated 5,6,7-trimethoxyindoles, therefore, copper-catalyzed
Ullmann-type N-arylation is the appropriate method of choice
in this study.
The structural analysis of [6,7,7]-tricyclic colchicine shows that
it also possesses a trimethoxyphenyl motif, thus attracting our
attention to the synthesis of [6,5]-bicyclic 5,6,7-trimethoxyindoles
aSchool of Pharmacy, College of Pharmacy, Taipei Medical University, 250
Wuxing Street, Taipei 11031, 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
† Electronic supplementary information (ESI) available: Spectral data of
compounds 6–12 and experimental procedures for synthesis and biological
evaluations, and HPLC purity data for compounds 6–12. See DOI:
10.1039/c0ob01038c
The synthetic route to obtain the desired target com-
pounds is as follows. A literature survey shows that the core
‡ These authors contributed equally to this work.
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5,6,7-trimethoxyindole (18) had been synthesized by R. D. Morin
et al. in 1957.12 R. D. Morin and colleagues utilized reductive
cyclization13 a common approach to the indole ring to accom-
plish the synthesis of 5,6,7-trimethoxyindole from 2-nitro-3,4,5-
trimethoxy-b-nitrostyrene in an overall yield of 4.6%. The low
synthetic yield is because the nitration of 3,4,5-trimethoxy-b-
nitrostyrene provided 2-nitro-3,4,5-trimethoxy-b-nitrostyrene in
9.3% yield only. In order to synthesize 5,6,7-trimethoxyindole
efficiently, the commercially available compound 13 was used
as a starting material (Scheme 1). The methyl ester of 13 was
hydrolyzed to afford the corresponding carboxylic acid 14 followed
by treatment with borane in boiling THF to furnish 15. The
primary alcohol 15 was oxidized by pyridinium dichromate to
provide the corresponding aldehyde 16. In order to achieve
reductive cyclization for indole formation, compound 16 was
treated with nitromethane in the presence of boiling acetic
acid to obtain 2-nitro-3,4,5-trimethoxy-b-nitrostyrene (17) in an
overall yield of 41.9% from starting material 13. Compared with
the literature, we have provided an efficient methodology to
achieve the synthesis of 2-nitro-3,4,5-trimethoxy-b-nitrostyrene
(17). Treatment of compound 17 with ferric powder in the presence
of acetic acid afforded 5,6,7-trimethoxyindole (18).
Scheme 2 Synthetic route to N-1 substituted 5,6,7-trimethoxyindoles
(6–12): a. substituted aryl bromide, CuO, K2CO3 or Cs2CO3, DMF, reflux
or microwave, 4.9%–9%; b. substituted benzyl chloride/bromide, KOH,
KI, DMF, 35%–40%.
oxide in boiling ethanol to provide 6–10. In addition, 5,6,7-
trimethoxyindole analogues connected with various aryl rings
with methylene linkage were investigated as well. Compound
18 was reacted with 4-methylbenzyl bromide and 3-fluoro-4-
methoxybenzyl chloride in the presence of KOH to yield 11 and
12, respectively.
Biological evaluation. A. in vitro cell growth inhibitory
activity
In an attempt to evaluate the effect of N-1 substitution on
the cancer cell inhibitory ability, the synthesized N-aryl-5,6,7-
trimethoxyindoles (6–10), and reference compounds CA4 and
colchicine were evaluated for their antiproliferative activities
against four human cancer cell lines, cervical carcinoma KB cells,
colorectal carcinoma HT29 cells, non-small-cell-lung carcinoma
H460 cells, and stomach carcinoma MKN45 cells (Table 1).
To examine whether 5,6,7-trimethoxyindole could provide an
equivalent effect of A-ring of CA4, compound 6 containing
3¢-hydroxy-4¢-methoxybenzene (B-ring of CA4) was generated.
In the antiproliferative evaluation, compound 6 demonstrated
substantial activity comparable to the reference compound CA4.
Compound 6 inhibited the growth of KB, HT29, MKN45 and
H460 cancer cell lines with IC50 values of 13.7, 10.2, 11.1
and 12.9 nM, respectively. Since the antiproliferative activity of
compound 6 is comparable to CA4, 5,6,7-trimethoxyindole could
Scheme 1 Synthetic route to 5,6,7-trimethoxyindole (18): a. KOH,
MeOH, reflux, 95%; b. BH3–THF, reflux, 80%; c. pyridinium dichromate,
molecular sieve, CH2Cl2, 65%; d. nitromethane, NH4OAc, AcOH, 100 ◦C,
85%; e. Fe powder, acetic acid, EtOH, 100 ◦C, 30%.
To explore the structure–activity relationship of the substituent
at the N-1 position, a series of N-aryl and heteroaryl-5,6,7-
trimethoxyindoles (6–12) were prepared as shown in Scheme 2.
To synthesize a series of N-heteroaryl-5,6,7-trimethoxyindoles,
compound 18 was treated with various aryl halides and copper
Table 1 IC50 values (nM SDa) of compounds 6–12, colchicine and CA4
Cell Type (IC50 SDa nM)
Compd
KB
HT29
10.2 3.1
3120.5 959.5
40.1 2.1
MKN45
11.1 3.6
1718.0 816.0
35.4 7.6
H460
12.9 5.2
4250.0 458.7
38.5 5.5
6
13.7 4.8
7
1226.5 125.2
48.9 45.0
> 5000
8
9
> 5000
> 5000
> 5000
10
31.6 10.9
946.3 234.0
751.7 31.8
2.0 0.4
50.1 10.6
964.0 62.2
770.5 35.1
715.1 32.6
15.9 4.9
38.6 9.5
1005.0 24.0
490.5 46.6
8.5 2.5
38.3 8.4
1096.2 316.5
870.0 89.1
19.0 3.5
11
12
CA4
Colchicine
10.3 0.9
17.9 1.8
17.0 5.1
a SD: standard deviation. All experiments were independently performed at least three times.
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Table 2 Inhibition of tubulin polymerization and colchicine binding by
compounds 6–10, colchicine and CA4
be considered to replace the core 3,4,5-trimethoxybenzene of CA4
revealing an avenue for future optimization.
In our previous studies of 3,4,5-trimethoxybenzoylindole6
and 3,4,5-trimethoxybenzoylquinoline derivatives,7 the attach-
ment of N-methylindole and quinoline moieties to 3,4,5-
trimethoxybenzoyl group resulted in an improved anticancer
activity. Accordingly, N-methylindole and quinoline groups were
selected and applied to the current study. Therefore, a series
of 5,6,7-trimethoxyindoles with N-methylindolyl and quinolinyl
groups were synthesized (7–10) as well. In the series of N-(N-
methylindolyl)-5,6,7-trimethoxyindoles, compound 8 exhibited
cancer cell inhibitory activity with IC50 values in the range of 35 to
50 nM. A comparison between compounds 7 and 8 demonstrated
that the N-methylindol-5-yl group of compound 8 contributed
to a 25 to 110 fold increase in cytotoxicity as compared to 7
with an N-methylindol-4-yl group. This phenomenon was also
observed when compounds 9 and 10 were compared. Compound
10 with a quinolin-6-yl group showed a 20 to 30 fold improved
cytotoxicity as compared to 9 with an N-methylindol-5-yl group
against several human cancer cell lines with IC50 values of 30 to
50 nM. This observation shows that the regiospecificity played a
significant role in improving the activity and the heteroatom at
the para position to N-1 nitrogen is preferred. Compounds 11
and 12 were synthesized to explore the effect of distance between
5,6,7-trimethoxyindole and substitutions. Compounds 11 and 12
exhibited weak activities against a panel of human cancer cell lines
as compared to compounds 6 to 10. This result indicated that the
methylene linkage between 5,6,7-trimethoxyindole and the aryl
substituent is detrimental to cytotoxic activity.
Compd
Tubulina IC50 SD (mM)
Colchicine bindingb (% SD)
6
2.5 0.2
> 10
2.5 0.3
> 10
82
77
78
95
2
—
3
—
5
7
8
9
10
3.2 0.2
3.9 0.4
1.7 0.2
Colchicine
CA4
2
a Inhibition of tubulin polymerization.14 b Inhibition of [3H] colchicine
binding.15 Tubulin was at 1 mM; both [3H]colchicine and inhibitor were at
5 mM.
B. Inhibition of tubulin polymerization and
colchicine binding activity
To investigate whether the activities of these N-aryl-5,6,7-
trimethoxyindoles were related to interactions with the mi-
crotubule system, compounds 6–10 and reference compounds
colchicine and CA4 were evaluated for their antitubulin activities
and colchicine binding activities (Table 2). The results indicated
that the compounds’ antiproliferative activity correlated with the
inhibition of tubulin polymerization. Compounds 6, 8 and 10 were
efficacious in inhibiting microtubulin assembly, with IC50 values of
2.5, 2.5 and 3.2 mM, respectively. These values were comparable to
reference compounds colchicine and CA4. In the [3H]colchicine-
binding assay, results demonstrated that 6, 8 and 10 were bound
to the colchicine binding site.
Fig. 2 Investigation of vascular disrupting activity (VDA) of compound
6. HUVECs were plated on Matrigel and allowed to form capillary tubes
in the presence of VEGF (20 ng mL-1) followed by exposure to different
concentrations of compound 6. Cultures were photographed and the
number of capillary tube networks was determined by counting under
a microscope (original magnification 100¥). Data reflect the mean number
of capillary tube networks relative to the to vehicle control group (DMSO)
standard deviation (SD) from three separate experiments.
Conclusions
In the analysis of several potent antimitotic natural products, the
trimethoxyphenyl is a crucial scaffold and attracts our attention for
optimization. Hence, a series of N-aryl-5,6,7-trimethoxyindoles
(6–12) were synthesized via copper-mediated Ullmann-type N-
arylation of 5,6,7-trimethoxyindole and investigated for cancer
cell growth inhibitory activities. Among the synthesized N-aryl-
5,6,7-trimethoxyindoles, compound 6 showed the best potency,
C. Investigation of vascular disrupting activity
In addition to tubulin polymerization inhibitory activity, CA4P (3)
has been shown to cause vascular shutdown within solid tumors
as vascular disrupting agents (VDA).16 Therefore, compound 6
which displayed the most potent antiproliferative activity was
investigated for vascular disrupting activity. The HUVECs were
plated on Matrigel and allowed to form capillary tubes in the
presence of VEGF (20 ng mL-1) followed by exposure to different
concentrations of compound 6.17 As shown in Fig. 2, compound
6 is capable of disrupting formed capillaries in a concentration-
dependent manner without affecting cell viability.
which is comparable to CA4, against a panel of cancer cells (IC50
=
10 to 14 nM). It also exhibited substantial inhibition of tubulin
polymerization with IC50 values of 2.5 mM which is comparable
to those of reference compounds as well. The vascular disrupting
activity assay indicated that compound 6 has a concentration-
dependent action on the HUVECs. The relative position of
the heteroatom to the N-1 nitrogen has significant impact on
the antiproliferative activity in the series of N-heteroaryl-5,6,
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7-trimethoxyindole analogues (7–10). The position of heteroatom
on substituents, N-methylindolyl and quinonlinyl, is preferred
to be para to the N-1 of 3,4,5-trimethoxyindole. In summary,
the modification from 3,4,5-trimethoxyphenyl (A-ring of CA4)
to 5,6,7-trimethoxyindole provides an opportunity for further
optimization of CA4 structure based anitproliferative agents.
7 C. Y. Nien, Y. C. Chen, C. C. Kuo, H. P. Hsieh, C. Y. Chang, J. S.
Wu, S. Y. Wu, J. P. Liou and J. Y. Chang, J. Med. Chem., 2010, 53,
2309.
8 (a) D. M. D’Souza and T. J. J. Mu¨ller, Chem. Soc. Rev., 2007, 36, 1095;
(b) J. J. Song, J. T. Reeves, F. Gallou, Z. Tan, N. K. Yee and C. H.
Senanayake, Chem. Soc. Rev., 2007, 36, 1120.
9 (a) R. Hersperger, K. B. French, L. Mazzoni and T. Mu¨ller, J. Med.
Chem., 2000, 43, 675; (b) H. C. Zhang, C. K. Derian, D. F. McComsey,
K. B. White, H. Ye, L. R. Hecker, J. Li, M. F. Addo, D. Croll, A. J.
Eckardt, C. E. Smith, Q. Li, W. M. Cheung, B. R. Conway, S. Emanuel,
K. T. Demarest, P. A. Gordon, B. P. Damiano and B. E. Maryanoff,
J. Med. Chem., 2005, 48, 1725.
Acknowledgements
This research was supported by the National Science Council,
Taiwan (grant no. NSC 99-2323-B-038-001, NSC 98-2113-M-038-
002-MY2), and the Department of Health, Taiwan (grant no.
DOH99-TD-C-111-004).
10 H. Xu, Mini-Rev. Org. Chem., 2009, 6, 367.
11 I. P. Beletskaya and A. V. Cheprakov, Coord. Chem. Rev., 2004, 248,
2337.
12 R. D. Morin, F. Benington and L. C. Clark, Jr., J. Org. Chem., 1957,
22, 331.
13 (a) P. D. Rege, Y. Tian and E. J. Corey, Org. Lett., 2006, 8, 3117; (b) K. C.
Nicolaou, U. Majumder, S. P. Roche and D. Y. K. Chen, Angew. Chem.,
Int. Ed., 2007, 46, 4715.
14 (a) J. P. Liou, K. S. Hsu, C. C. Kuo, C. Y. Chang and J. Y. Chang,
J. Pharmacol. Exp. Ther., 2007, 323, 398; (b) J. Y. Chang, M. J. Lai,
Y. T. Chang, H. Y. Lee, Y. C. Cheng, C. C. Kuo, M. C. Su, C. Y. Chang
and J. P. Liou, Med. Chem. Commun., 2010, 1, 152.
Notes and references
1 (a) C. Dumontet and M. A. Jordan, Nat. Rev. Drug Discovery, 2010, 9,
790; (b) A. Jordan and L. Wilson, Nat. Rev. Cancer, 2004, 4, 253.
2 (a) C. M. Lin, S. B. Singh, P. S. Chu, R. O. Dempcy, J. M. Schmidt,
G. R. Pettit and E. Hamel, Mol. Pharmacol., 1988, 34, 200; (b) C. M.
Lin, H. H. Ho, G. R. Pettit and E. Hamel, Biochemistry, 1989, 28, 6984.
3 (a) G. C. Tron, T. Pirali, G. Sorba, F. Pagliai, S. Busacca and A. A.
Genazzani, J. Med. Chem., 2006, 49, 3033; (b) H. P. Hsieh, J. P. Liou
and N. Mahindroo, Curr. Pharm. Des., 2005, 11, 1655; (c) Q. Li and
H. L. Sham, Expert Opin. Ther. Pat., 2002, 12, 1663; (d) N. Mahindroo,
J. P. Liou and J. Y. Chang, Expert Opin. Ther. Pat., 2006, 16, 647;
(e) D. J. Chaplin, M. R. Horsman and D. W. Siemann, Curr. Opin.
Investig. Drugs, 2006, 7, 522.
4 G. R. Pettit, C. J. Temple, V. L. Narayanan, R. Varma, M. J. Simpson,
M. R. Boyd, G. A. Rener and N. Bansal, Anti-cancer Drug Des., 1995,
10, 299.
5 K. Ohsumi, R. Nakagawa, Y. Fukuda, T. Hatanaka, Y. Morinaga, Y.
Nihei, K. Ohishi, Y. Suga, Y. Akiyama and T. Tsuji, J. Med. Chem.,
1998, 41, 3022.
15 C. C. Kuo, H. P. Hsieh, W. Y. Pan, C. P. Chen, J. P. Liou, S. J. Lee, Y. L.
Chang, L. T. Chen and J. Y. Chang, Cancer Res., 2004, 64, 4621.
16 (a) G. G. Dark, S. A. Hill, V. E. Prise, G. M. Tozer, G. R. Pettit and
D. J. Chaplin, Cancer Res., 1997, 57, 1829; (b) D. W. Siemann, M. C.
Bibby, G. G. Dark, A. P. Dicker, F. A. L. M. Eskens, M. R. Horsman,
D. Marme and P. M. LoRusso, Clinical Cancer Res., 2005, 11, 416;
(c) G. M. Tozer, C. Kanthou and B. C. Baguley, Nat. Rev. Cancer,
2005, 5, 423; (d) D. M. Patterson and G. J. S. Rustin, Clin. Oncol., 2007,
19, 443; (e) G. M. Tozer, S. Akerman, N. A. Cross, P. R. Barber, M. A.
Bjorndahl, O. Greco, S. Harris, S. A. Hill, D. J. Honess, C. R. Ireson,
K. L. Pettyjohn, V. E. Prise and C. C. Reyes-Aldasoro, Cancer Res.,
2008, 68, 2301; (f) D. W. Siemann, D. J. Chaplin and P. A. Walicke,
Expert Opin. Invest. Drugs, 2009, 18, 189.
¨
17 G. Kremmidiotis, A. F. Leske, T. C. Lavranos, D. Beaumont, J. Gasic,
A. Hall, M. O’Callaghan, C. A. Matthews and B. Flynn, Mol. Cancer
Ther., 2010, 9, 1562.
6 J. P. Liou, C. Y. Wu, H. P. Hsieh, C. Y. Chang, C. M. Chen, C. C. Kuo
and J. Y. Chang, J. Med. Chem., 2007, 50, 4548.
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