One-pot synthesis of benzo[b]furan and indole inhibitors of tubulin polymerization

Article abstract of DOI:10.1021/jm020077t

Benzo[b]furan and indole analogues of some recently identified benzo[b]thiophene inhibitors of tubulin polymerization have been prepared, and their biological activity has been assessed. Several very potent analogues were identified.

Full text of DOI:10.1021/jm020077t

2670
J . Med. Chem. 2002, 45, 2670-2673
On e-P ot Syn th esis of Ben zo[b]fu r a n a n d In d ole In h ibitor s of Tu bu lin
P olym er iza tion
Bernard L. Flynn,*,† Ernest Hamel,‡ and M. Katherine J ung§
Department of Chemistry, The Faculties, Australian National University, Canberra, ACT, 0200, Australia,
Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, and Science Applications
International CorporationsFrederick, National Cancer Institute at Frederick, National Institutes of Health,
Frederick, Maryland 21702
Received February 14, 2002
Benzo[b]furan and indole analogues of some recently identified benzo[b]thiophene inhibitors
of tubulin polymerization have been prepared, and their biological activity has been assessed.
Several very potent analogues were identified.
Compounds that interfere with the microtubule-
tubulin equilibria in cells are useful in the treatment
of human disease.1 Examples are the antiinflammatory
activity of colchicine,1a the anthelmintic properties of
the benzimidazole carbamates,1b and the antineoplastic
use of the Catharanthus (formerly Vinca) alkaloids1c,d
and the taxoids.1e,f More recently, it has been estab-
lished that some tubulin binders selectively target the
vascular system of tumors.2 These agents induce mor-
F igu r e 1.
phological changes in the endothelial cells of the tumor’s
blood vessels so as to occlude flow.2i As a result, virtually
complete vascular shutdown is achieved within the
tumor in a matter of minutes. Combretastatin A-4 (CA4,
Figure 1) is a powerful inhibitor of tubulin polymeriza-
tion and a potent cytotoxin (Table 1).3 The prodrug form
of CA4, combretastatin A-4 disodium phosphate (CA4P),
is undergoing clinical trials as a tumor vascular target-
ing agent.2 The prodrug, CA4P, is itself inactive with
purified tubulin and is therefore believed to require in
vivo dephosphorylation to CA4 before it can act.2 This
process may contribute to its selectivity, with greater
rates of dephosphorylation occurring in the tumor
vascular systems than in normal vasculature.2c,j
method to the preparation of various benzo[b]furan and
indole analogues of 1 and 2 and their effects on tubulin
polymerization.
Benzo[b]furans 8 and 9 were prepared by multicom-
ponent coupling of 2-iodo-5-methoxyphenol 3, 4-meth-
Sch em e 1a
The success of tubulin polymerization inhibitors as
anticancer agents has stimulated significant interest in
the identification of new compounds that may be more
potent or more selective in targeted tissues or tumors.
In 1999, Pinney and co-workers4 reported a benzo[b]-
thiophene, compound 1 (Figure 1), that exhibited some
activity as a tubulin polymerization inhibitor and ap-
peared to bind weakly to the colchicine binding domain
of tubulin. However, 1 only inhibited the rate and not
the extent of tubulin polymerization. The limited activ-
ity of 1 was hypothesized to come from poor solubility.4a
Using our novel method for the flexible and concise
synthesis of benzo[b]thiophenes, we prepared analogues
of 1.5 The most potent of these was 2 (Figure 1, Table
1).5a More recently, we have developed a one-pot,
multicomponent coupling approach to benzo[b]furans
and indoles.6 Here, we report on the application of this
* To whom correspondence should be addressed. Phone: 61 2 6125
2017. Fax: ++61 2 6125 0760. E-mail: flynn@rsc.anu.edu.au.
† Australian National University.
a
Reagents and conditions: (i) MeMgCl 2 equiv, Pd(PPh3)2Cl2
3 mol %, THF, 65 °C, 1.5 h under N2(g); (ii) cool to room temp,
add 7 and DMSO, then heat to 80 °C, 16-18 h; (iii) same as (ii)
except exchange N2(g) atmosphere for CO(g) atmosphere (balloon);
(iv) AlCl3 3 equiv, CH2Cl2.
‡ Division of Cancer Treatment and Diagnosis, National Cancer
Institute at Frederick, National Institutes of Health.
§ Science Applications International CorporationsFrederick, Na-
tional Cancer Institute at Frederick, National Institutes of Health.
10.1021/jm020077t CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/04/2002

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2671
Ta ble 1. Effects of Indole and Benzo[b]furan Compounds on Tubulin Polymerization, Colchicine Binding, and Growth of MCF-7
Human Breast Carcinoma Cells8
inhibition of tubulin
inhibition of colchicine bindingb
inhibition of cell growth,c
compound
X
Y
R
polymerization,a IC50 ( SD (µM)
(% inhibition ( SD)
IC50 ( SD (nM)
CA4
1
2
8
9
11
13
17
19
20
21
22
2.1 ( 0.1d
91 ( 2
11 ( 4d
S
S
O
O
CdO
CdO
nothing
CdO
CdO
nothing
nothing
CdO
CH(OH)
CH(OH)
CH2
H
OH
H
H
OH
H
OH
OH
H
OH
H
>40e,f
640 ( 10d
520 ( 400d
>1000
3.4 ( 0.2d
>40
21 ( 10d
0.41 ( 0.1
1.3 ( 0.2
>40e
77 ( 3
80 ( 2
34 ( 10
42 ( 10
460 ( 5
370 ( 2
45 ( 5
>1000
560 ( 60
400 ( 100
O
NH
NH
NH
O
O
O
4.1 ( 0.6
28 ( 8
54 ( 0.7
1.6g
>40e
8.8 ( 1.4
2.5 ( 0.3
17 ( 10
a
The tubulin concentration was 10 µM. Inhibition of extent of assembly, after a 20 min incubation at 30 °C, was the parameter measured.
The tubulin concentration was 1.0 µM, and the [3H]colchicine and inhibitor concentrations were both 5.0 µM. Incubation was for 10 min
b
at 37 °C. At least two independent experiments were performed with each of the compounds evaluated. c Cells were grown for 48 h at 37
°C in a humidified 5% CO2 atmosphere. Cell protein was the parameter measured. At least two independent experiments were performed
d
with each compound. Data from ref 5. e The rate, but not the extent of assembly, was reduced by compound concentrations as high as
g
40 µM. f Data from ref 4a. Same value in two experiments.
Sch em e 2a
oxyphenylethyne 4, and 3,4,5-trimethoxyiodobenzene 7
(Scheme 1).6 This process involved initial deprotonation
of 3 and 4 with 2 equiv of methylmagnesium chloride
to give the corresponding magnesium phenolate and
acetylide, respectively (not shown). Coupling occurred
upon addition of a catalytic amount of Pd(PPh3)2Cl2 and
heating to 65 °C for 1.5 h to give the intermediate
o-alkynylphenolate 6. The solution was diluted with
dimethyl sulfoxide (DMSO), aryl iodide 7 was added,
and the reaction was heated to 80 °C for 16 h under
N2(g). Under these conditions, the palladium(0) present
undergoes oxidative addition to 7, followed by palla-
dium(II)-induced cyclization of 6 and reductive elimina-
tion to afford 8 in an excellent yield (88%). When the
N2(g) atmosphere was exchanged for one of CO(g) at the
point of introduction of 7, a carbonyl is inserted between
a
Reagents and conditions: (i) Pd(PPh3)2Cl2 3 mol %, Et3N 2
palladium(II) and the trimethoxyphenyl group, leading
equiv, CuI 6 mol %, CH3CN, 18 °C, 1 h under N2(g); (ii) K2CO3
5
to the carbonylated product 9 (64%).6 Using alkyne 5
in place of 4 under these carbonylative conditions gave
10 (63%). The isopropyl ether in 10 was cleaved using
AlCl3 to give the desired phenol 11 (97%).7
equiv, 7, 18 °C, 18 h; (iii) same as (ii) except exchange N2(g)
atmosphere for CO(g) atmosphere (balloon); (iv) AlCl3 4 equiv,
CH2Cl2.
the o-iodotrifluoroacetanilide 14 was coupled to alkyne
5 under Sonogashira conditions in acetonitrile to give
15 (Scheme 2). When this coupling was complete (ap-
proximately 2 h), the aryl iodide 7 and 5 equiv of K2-
CO3 were added and the solution was left stirring for a
further 18 h to give 16 (77%). As before, when the N2-
(g) atmosphere was exchanged for one of CO(g) at the
point of introduction of 7, the product obtained was the
carbonylated product 18 (73%). Both 16 and 18 were
deprotected to the corresponding phenols 17 and 19,
respectively, using AlCl3.7,10,11 These alternative mul-
ticomponent coupling conditions are not applicable to
the synthesis of 2,3-disubstituted benzo[b]furans be-
cause, under the Sonogashira conditions of coupling, the
initially formed o-alkynylphenols rapidly cyclize to the
simple C3-unsubstituted benzo[b]furans.6
Indoles could be prepared by using o-iodoacetanilides
in place of the o-iodophenols in the multicomponent
coupling reaction.6 Accordingly, reaction of 12 with 4
and 7, under the multicomponent coupling conditions
described above, gave 13 (85%) (eq 1).6 The acyl group
was lost during the course of the reaction.6
Alternatively, indoles could be obtained at room
temperature in a one-pot reaction using a variant of the
stepwise sequence described by Cacchi.9 Accordingly,
Additional analogues were generated by reduction of
ketones 9 and 11 to give alcohols 20 and 21, respec-

2672 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Brief Articles
Sch em e 3a
was supported in part by NCI, National Institutes of
Health Contract N01-CO-1240.
Su p p or tin g In for m a tion Ava ila ble: Procedures for the
synthesis of all new compounds in Table 1, including spectro-
scopic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
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clinical review. Br. J . Cancer 2000, 82, 1907-1913. (e) Rowinsky,
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a
Reagents and conditions: (i) NaBH4 2 equiv, EtOH; (ii)
CF3CO2H 10 equiv, Et3SiH 5 equiv, CH2Cl2.
tively, using sodium borohydride (Scheme 3). Alcohol
20 was further reduced to the simple methylene system
22 by reaction with trifluoroacetic acid and triethyl-
silane in excellent yield.
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Compounds 8, 9, 11, 13, 17, 19, and 20-22 were
evaluated for inhibition of tubulin assembly (Table 1).
Those that displayed a significant inhibitory effect
(defined as IC50 < 5.0 µM) were also examined for an
inhibitory effect on the binding of [3H]colchicine to
tubulin (the tubulin used in these studies was purified
to electrophoretic homogeneity from bovine brain).
These compounds were also evaluated for cytotoxicity
against MCF-7 human breast carcinoma cells (Table 1).
Compound 8 did not inhibit tubulin assembly at
concentrations as high as 40 µM or cell growth at 1 µM
and was not further examined, while compounds 13, 17,
20, and 21 showed moderate to poor activity both as
tubulin polymerization inhibitors and as cytotoxins.
Compounds 9, 11, 19, and 22 were all similar to or more
potent than CA4 as inhibitors of tubulin polymerization.
Compound 9 was the most active, exhibiting 5 times the
level of potency of CA4. However, all of these compounds
(9, 11, 19, and 22) were less potent than CA4 as
inhibitors of [3H]colchicine binding to tubulin and as
cytotoxins (against MCF-7 human breast carcinoma
cells). It is nonetheless noteworthy that the cytotoxicity
of 9, 11, and 19 was only slightly less than that of CA4.
In terms of a structure-activity relationship (SAR)
for benzofused heterocyclic compounds, those containing
either a nitrogen or an oxygen in the heterocyclic ring
are much more effective than their sulfur analogues in
all three biological assays (compare 1 and 2 with 9, 11,
and 19). As had been previously demonstrated for the
sulfur series, the absence of a one-carbon linker between
C3 of the benzofused heterocycle and the trimethox-
yphenyl ring leads to a complete or near-complete loss
in activity (compare 8, 13, and 17 with 9, 11, and 19).5
This carbon linker is much more effective as a carbonyl
group than as a carbinol or simple methylene group
(compare 9 and 11 with 20-22).
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Con clu sion
Multicomponent coupling of o-iodophenols (or o-
iodoacetanilides) with terminal alkynes and aryl iodides
provides rapid access to potent benzo[b]furan- and
indole-based tubulin polymerization inhibitors. These
systems represent valuable new leads in the pursuit of
anticancer chemotherapies.
(5) (a) Flynn, B. L.; Verdier-Pinard, P.; Hamel, E. A novel palladium-
mediated coupling approach to 2,3-disubstituted benzo[b]-
thiophenes and its application to the synthesis of tubulin binding
agents. Org. Lett. 2001, 3, 651-654. (b) Flynn, B. L.; Flynn, G.
P.; Hamel, E.; J ung, M. K. The synthesis and tubulin binding
activity of thiophene-based analogues of combretastatin A-4.
Bioorg. Med. Chem. Lett. 2001, 11, 2341-2343.
Ack n ow led gm en t. The authors thank the Austra-
lian Research Council for financial support, including
an Australian Research Fellowship to B.L.F. This work

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2673
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J M020077T