Synthesis and preliminary biological evaluation of new anti-tubulin agents containing different benzoheterocycles

Article abstract of DOI:10.1016/j.bmcl.2005.06.022

A new series of compounds, in which the 2-amino-4-methoxyphenyl ring of phenstatin analogue 5 was replaced with 2- or 3-amino-benzoheterocycles, was synthesized and evaluated for antiproliferative activity and inhibition of colchicine binding. The lack of

Full text of DOI:10.1016/j.bmcl.2005.06.022

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4048–4052
Synthesis and preliminary biological evaluation of new
anti-tubulin agents containing different benzoheterocycles
Romeo Romagnoli,a,* Pier Giovanni Baraldi,a M. Katherine Jung,b
Maria Antonietta Iaconinoto,a Maria Dora Carrion,a Vincent Remusat,a
Delia Preti,a Mojgan Aghazadeh Tabrizi,a Fruttarolo Francesca,a
Erik De Clercq,c Jan Balzarinic and Ernest Hameld
a
´
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, Via Fossato di Mortara 17/19, 44100 Ferrara, Italy
bScience Applications International Corporation–Frederick Inc., National Cancer Institute at Frederick,
National Institutes of Health, Frederick, MD 21702, USA
cRega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Minderbroedersstraat 10, B-3000 Leuven, Belgium
dScreening Technologies Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, MD 21702, USA
Received 7 May 2005; revised 1 June 2005; accepted 6 June 2005
Available online 7 July 2005
Abstract—A new series of compounds, in which the 2-amino-4-methoxyphenyl ring of phenstatin analogue 5 was replaced with 2- or
3-amino-benzoheterocycles, was synthesized and evaluated for antiproliferative activity and inhibition of colchicine binding. The
lack of activity of 30,40-dimethoxy- and 40-methoxy-benzoyl derivatives (8 and 9, respectively) indicates that the 30,40,50-tri-
methoxybenzoyl moiety is critical for the activity. Two compounds, 7 and 11, displayed potent antiproliferative activity, with
IC50 values ranging from 25 to 100 nM against a variety of cancer cell lines. Derivative 11 was more active than CA-4 as an inhibitor
of tubulin polymerization. The results demonstrated that the antiproliferative activity was correlated with inhibition of tubulin
polymerization.
Ó 2005 Elsevier Ltd. All rights reserved.
Despite the progress made in recent years in the sector
of new drugs endowed with antiangiogenic activity, this
sector of research is still considered as one of the most
promising fields for the discovery of new drugs for the
treatment of diseases characterized by abnormal angio-
genesis, and in particular for the treatment of tumors.1
Research oriented toward the discovery of new genera-
tion agents useful in cancer chemotherapy has identified
tubulin as a possible cellular target.2a
sequently, natural and synthetic substances capable of
altering the polymerization or depolymerization of
microtubules have been effective as chemotherapeutic
agents.2b Combretastatin A-4 (CA-4, 1), isolated from
an African willow, Combretum caffrum (Combreta-
ceae),3 shows interesting anticancer potential due to
its antitubulin properties. CA-4 strongly binds to the
colchicine site of tubulin.4 This binding prevents tubu-
lin polymerization and causes an antimitotic effect.
CA-4 inhibits cell growth at low to mid-nanomolar
concentrations.3 The sodium phosphate prodrug of
CA-4 (CA-4P, 2) is water-soluble and has yielded
promising results in current Phase I human cancer
clinical trials.5 Its structural simplicity, along with the
ability to selectively damage tumor neovascularization,
makes CA-4 of great interest from the medicinal chem-
istry viewpoint.
The microtubule cytoskeleton plays a very important
role in regulating cell architecture. The microtubule
systems of eukaryotic cells comprise a dynamic matrix
in which heterodimers of tubulin polymerize to form
microtubules both in neoplastic and normal cells. Con-
Keywords: Anti-tubulin agents; Benzoheterocycles; Combretastatin-
A4.
For these reasons, a large number of CA-4 analogues
have been synthesized and evaluated in structure–activ-
ity relationship (SAR) studies.6 Replacing the ethene
*
Corresponding author. Tel.: +39 0532 291290; fax: +39 0532
291296; e-mail: rmr@unife.it
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.022

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4048–4052
4049
bridge of CA-4 with a carbonyl group furnished a ben-
zophenone derivative named Phenstatin (3), which was
found to be a very strong cytotoxic agent with the same
characteristics as CA-4.7 Changing the position of the
hydroxyl group from position C-3 to C-2, to furnish
the Phenstatin isomer 4, dramatically decreased bioac-
tivity.8 Replacing the hydroxyl moiety at the C-2 posi-
tion of the benzophenone ring (compound 5) with an
amino group increased cytotoxicity 100-fold as com-
pared with 4, indicating that the amino and hydroxy
groups were not bioequivalent at the C-2 position. The
2-amino benzophenone derivative 5 showed significantly
increased cytotoxicity against many human cancer cell
lines as compared with Phenstatin 3, but 5 was neverthe-
less slightly less potent than CA-4.8 Lack of the methoxy
group (compound 6) at the C-4 position resulted in sig-
nificantly decreased growth inhibitory activity.
Compounds 7–9 were synthesized by a four-step synthe-
sis described in Scheme 1.11 2-Amino-3-aroyl 4,5,6,7-tet-
rahydrobenzo[b]thiophenes 20–22 were obtained by the
Gewald reaction12 applied to b-ketonitriles 17–1913
and cyclohexanone. Acetylation of the amino group
using a mixture of acetic anhydride and pyridine, and
the subsequent dehydrogenation with Pd/C with heating
afforded the benzo[b]thiophene derivatives 23–25, which
were transformed by saponification into the final prod-
ucts 7–9.
The two ligands based on the 3-amino-benzo[b]thio-
phene molecular skeleton (10 and 11), along with the
3-amino-benzo[b]furan 12, were prepared in excellent
yield by a Ôone-potÕ cyclization reaction between 2-cya-
nothiophenol 26, 4-methyl-2-cyanothiophenol 27, and
2-cyanophenol 28, respectively, with 2-bromo-30,40,50-
trimethoxyacetophenone 2914 and K2CO3 in acetone
(Scheme 2). Compounds 26 and 27 were synthesized
by the condensation of commercially available 2-nitro-
benzonitrile 30 and 2-nitro-4-methylbenzonitrile 31,
respectively, with benzylmercaptan anion and subse-
quent S-debenzylation with aluminium chloride.15
In this article, we report the synthesis and biological
evaluation of new compounds 7–13, that are structurally
related to the 2-aminobenzophenones 5 and 6, in which
the 2-aminophenyl moieties of the latter derivatives were
replaced with various 2- or 3-aminobenzoheterocycles
such as benzo[b]thiophene, benzo[b]furan, and indole.
We confirmed that a number of these new compounds
are active cytotoxic agents with significant antitubulin
activity. Since the removal of either the C0-4 or the C0-
5 methoxy group causes a substantial loss of cytotoxicity
in the CA-4 system,9 through the synthesis of com-
pounds 8 and 9, we investigated the effect of the
30,40,50-trimethoxybenzoyl group in the 2-amino
benzo[b]thiophene derivative 7 on the antiproliferative
activity. The 3-(30,40,50-trimethoxybenzoyl)-indole, -benzo-
furan, and -benzothiophene molecular skeletons are the
core structure of a series of antitubulin agents (com-
pounds 14–16, respectively), which showed activity com-
parable to that of CA-4.10
Compound 13 was prepared following the procedure
reported in Scheme 3. Derivative 3216 was reacted with
bromoacetophenone 29 to give the N-ethoxycarbonyl
indole intermediate, which was converted into the corre-
sponding N-unsubstituted indole 13 after alkaline
hydrolysis.
In Table 1, we report the in vitro antiproliferative activ-
ity of compounds 7–13 and 20 against a panel of tumor
cell lines, using CA-4 as reference compound. It is note-
worthy that the antiproliferative effects of 7–8, 10–11,
and 20 were more pronounced against Molt/4 and
CEM as compared with L1210 and FM3A. Compounds

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R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4048–4052
Scheme 1. Reagents and conditions: (a) cyclohexanone, S8, morpholine, EtOH, 70 °C for 1 h then 18 h at rt; (b) Ac2O, pyridine, reflux; (c) 10% Pd/C
moistened with a 50% water, 130 °C, 18 h; (d) KOH, EtOH, reflux, 2 h.
Scheme 2. Reagents and conditions: (a) C6H5CH2SH, KOH, DMF; (b) AlCl3, C6H6; (c) K2CO3, acetone, reflux, 12 h.
Scheme 3. Reagents and conditions: (a) NaH, DMF, rt, 24 h; (b) NaOH, EtOH/H2O, reflux, 1 h.
7 and 11 demonstrated substantial growth-inhibitory ef-
fects on the proliferation of murine leukemia (L1210),
murine mammary carcinoma (FM3A), and human
T-lymphoblastoid (Molt/4 and CEM) cells. However,
CA-4 was more potent than any of the new compounds
in all cell lines examined.
Comparing the molecules which possess the trimethoxy
benzoyl moiety, the benzofused heterocyclic compounds
containing a nitrogen (13) or an oxygen (12) atom in the
heterocyclic ring are much less effective than their sulfur
analogues 7 and 10. Comparing the two benzo[b]thio-
phene isomers 7 and 10, the latter was about 4-fold less

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4048–4052
4051
Table 1. In vitro growth inhibition activity of compounds 7–13, 20, and CA-4 against murine leukemia (L1210), murine mammary carcinoma
(FM3A), and human T-lymphoblast (Molt/4 and CEM) cells
a
IC50 (nM)
Compound
L1210
FM3A
Molt4/C8
73
CEM
7
20
90 3.2
2240 122
3700 60
>10,000
350 320
58 50
100
0
9
74 15
5680 315
4700 2500
>10,000
1580 135
1700 200
9200 200
290 10
2150 234
1700 500
>10,000
8
9
10
1800 300
5
310 20
2
11
12
71
34
3
24
>10,000
>10,000
2.8 1.1
>10,000
>10,000
42 6.0
>10,000
>10,000
16 1.4
>10,000
>10,000
1.9 1.6
13
CA-4 (1)
Data are expressed as means SE from the dose–response curves of at least three independent experiments.
a IC50, compound concentration required to inhibit tumor cell proliferation by 50%.
Table 2. Inhibition of tubulin polymerization and colchicine binding
by compounds 7, 10, 11, and 20
active than 7 with L1210, Molt/4, and CEM cells, and
almost 20-fold less active with FM3A cells. The reduced
antiproliferative activity of the 4,5,6,7-tetrahydro-
benzo[b]thiophene derivative 20 as compared with 7
demonstrates that the aromaticity of the benzene ring
fused with the thiophene is critical for activity.
Compound
Tubulin assemblya
Colchicine bindingb
IC50 SD (lM)
%
SD
5 lM drug
2 lM drug
7
20
3.1 0.3
>40
57
3
1
ND
ND
ND
77
0.3
9
The series of derivatives 7–9 demonstrates that all three
methoxy groups are essential for activity. Substitution
of the trimethoxybenzene group with 30,40-di-
methoxybenzoyl and 40-methoxybenzoyl moieties (com-
pounds 8 and 9, respectively), almost eliminated growth
inhibition activity.
10
11
4.2 0.7
1.3 0.8
2.0 0.3
39
88
97
9
4
3
CA-4 (1)
6
95
ND, not determined.
a Inhibition of tubulin polymerization. Tubulin was at 10 lM.
b Inhibition of [3H]colchicine binding. Tubulin was at 1 lM, [3H]col-
chicine at 5 lM.
Compound 10, with a 3-(30,40,50-trimethoxybenzoyl)-2-
amino-benzo[b]thiophene nucleus, had moderate anti-
proliferative effects on the growth of L1210, Molt4,
and CEM cells (IC50 = 290–350 nM) and about 5-fold
lower activity against the FM3A cell line. The introduc-
tion of a lipophilic and electron-releasing methyl group
at the C-6 position of 10, to afford the derivative 11,
resulted in an increase in the antitumor activity by one
order of magnitude. This compound was especially effec-
tive against CEM and Molt4/C8 cells, with IC50Õs of 24
and 34 nM, respectively.
activity as cytotoxic agents, except that CA-4 was more
cytotoxic than compound 11.
When inhibitory effects on colchicine binding were eval-
uated, however, CA-4 proved to be somewhat more po-
tent than compound 11, which was the most potent of
the new agents, but otherwise order of activity in the
two tubulin-based assays was identical.
In conclusion, we have discovered a new type of inhibi-
tor of tubulin polymerization based on the 2-(30,40,50-
trimethoxybenzoyl)-3-amino-benzo[b]thiophene molec-
ular skeleton. Thus far the promising compound in this
series is the 2-(30,40,50-trimethoxybenzoyl)-3-amino-6-
methylbenzo[b]thiophene derivative 11. Compound 11
is a potent antiproliferative agent and inhibitor of tubu-
lin polymerization through binding to the colchicine-
binding site of tubulin. A noteworthy point was that
the preparation of 11 was carried out via an efficient
synthesis and it represents the lead compound of an
interesting new class of antitubulin agents with potential
utility for the treatment of human cancer.
To investigate whether the antiproliferative activities of
these compounds were related to an interaction with
tubulin, compounds 7, 10, 11, and 20 were evaluated
for inhibition of the polymerization of purified tubu-
lin.17 The same compounds were also examined for
inhibitory effects on the binding of [3H]colchicine to
tubulin18 (Table 2).
Compound 20 did not greatly alter tubulin assembly at
concentrations as high as 40 lM nor did it inhibit colchi-
cine binding to tubulin. Compounds 7, 10, and 11 all
strongly inhibited tubulin assembly, and compound 11
seemed to be even more active than the reference com-
pound CA-4. Compound
7 was half as active
(IC50 = 3.1 lM) and 10 about one-third as active
(IC50 = 4.2 lM) as 11. Thus, the order of inhibitory
effects on tubulin polymerization was 11 > CA-
4 > 7 > 10 >> 20. This order of activity as inhibitors of
tubulin assembly correlates well with their order of
Acknowledgment
This work was supported in part by NCI, National
Institutes of Health Contract # NO1-CO-12400.

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Lett. 1999, 9, 2303; compound 15: Flynn, B.; Hamel, E.;
References and notes
Jung, M. K. J. Med. Chem. 2002, 45, 2670; compound 16:
Pinney, K. G.; Bounds, A. D.; Dingeman, K. M.;
Mocharla, V. P.; Pettit, G. R.; Bai, R.; Hamel, E. Bioorg.
Med. Chem. Lett. 1999, 9, 1081.
1. Gourley, M.; Williamson, J. S. Curr. Pharm. Des. 2000, 6,
417.
2. (a) Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown,
A. T. Med. Res. Rev. 1998, 18, 259; (b) Rowinsky, E. K.;
Donehower, R. C. Pharmacol. Ther. 1992, 52, 35.
3. Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts,
D. S.; Garcia-Kendall, D. Experentia 1989, 45, 209.
4. Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E.
Biochemistry 1989, 28, 6984.
5. Pettit, G. R.; Temple, C., Jr.; Narayanan, V. L.; Varma,
R.; Boyd, M. R.; Rener, G. A.; Bansal, N. Anti-Cancer
Drug Des. 1995, 10, 299.
11. Baba, A.; Mori, A.; Yasuma, T.; Unno, S.; Makino, H.;
Sohda, T. Chem. Pharm. Bull. 1999, 47, 993.
12. Gewald, K.; Schinke, E.; Bo¨ettcher, H. Chem. Ber. 1966,
99, 94.
13. For the synthesis of 17, see: Seneci, P.; Nicola, M.; Inglesi,
M.; Vanotti, E.; Resnati, T. Synth. Commun. 1999, 29,
311, compound 18 was synthesized following the proce-
dure reported in Ref. 11; compound 19 was commercially
available.
14. Fujii, T.; Yoshifuji, S.; Ohba, M. Chem. Pharm. Bull.
1978, 26, 3218.
15. (a) Beck, J. R. J. Heterocyclic Chem. 1978, 15, 513;
(b) Carrington, D. E. L.; Clarke, K.; Scrowston, R. M.
J. Chem. Soc. 1971, 3262.
16. Radl, S.; Hezky, P.; Urbankova, J.; Vachal, P.; Krejci, I.
Collect. Czech Chem. Commun. 2000, 65, 280.
17. Hamel, E. Cell Biochem. Biophys. 2003, 38, 1.
18. Verdier-Pinard, P.; Lai, J.-Y.; Yoo, H.-D.; Yu, J.;
6. Nam, N. H. Curr. Med. Chem. 2003, 10, 1697.
7. Pettit, G. R.; Toki, B.; Herald, D. L.; Verdier-Pinard, P.;
Boyd, M. R.; Hamel, E.; Pettit, R. K. J. Med. Chem. 1998,
41, 1688.
8. Liou, J. P.; Chang, C. W.; Song, J. W.; Yang, Y. N.; Yeh,
C. F.; Tseng, H. Y.; Lo, Y. K.; Chang, Y. L.; Chang, C.
M.; Hsieh, H. P. J. Med. Chem. 2002, 45, 2556.
9. Cushman, M.; Nagarathnam, D.; Gopal, D.; Lin, C. M.;
Hamel, E. J. Med. Chem. 1992, 35, 2293.
´
Marquez, B.; Nagle, D. G.; Nambu, M.; White, J. D.;
10. For compound 14, see: Medarde, M.; Ramos, A.; Cabal-
´
lero, E.; Pelaez-Lamamiede Clairac, R.; Lopez, J. L.;
Gravalos, D. G.; Feliciano, A. S. Bioorg. Med. Chem.
Falck, J. R.; Gerwick, W. H.; Day, B. W.; Hamel, E. Mol.
Pharmacol. 1998, 53, 62.