Design, synthesis and structure-activity relationship of 2-(3′,4′,5′-trimethoxybenzoyl)-benzo[b]furan derivatives as a novel class of inhibitors of tubulin polymerization

Article abstract of DOI:10.1016/j.bmc.2009.08.027

The biological importance of microtubules in mitosis and cell division makes them an interesting target for the development of anticancer agents. Small molecules such as benzo[b]furans are attractive as inhibitors of tubulin polymerization. Thus, a new cl

Full text of DOI:10.1016/j.bmc.2009.08.027

Bioorganic & Medicinal Chemistry 17 (2009) 6862–6871
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and structure–activity relationship of
2-(30,40,50-trimethoxybenzoyl)-benzo[b]furan derivatives as a novel class
of inhibitors of tubulin polymerization
a,
a
a
a
Romeo Romagnoli a, , Pier Giovanni Baraldi , Maria Dora Carrion , Carlota Lopez Cara , Olga Cruz-Lopez ,
Manlio Tolomeo b, Stefania Grimaudo b, Antonietta Di Cristina b, Maria Rosaria Pipitone b, Jan Balzarini c,
Nicola Zonta d, Andrea Brancale d, Ernest Hamel e
*
*
a Dipartimento di Scienze Farmaceutiche, Università di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy
b Dipartimento Biomedico di Medicina Interna e Specialistica, Università di Palermo, Palermo, Italy
c Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Minderbroedersstraat 10, B-3000 Leuven, Belgium
d The Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3XF, UK
e Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The biological importance of microtubules in mitosis and cell division makes them an interesting target
for the development of anticancer agents. Small molecules such as benzo[b]furans are attractive as inhib-
itors of tubulin polymerization. Thus, a new class of inhibitors of tubulin polymerization based on the 2-
(30,40,50-trimethoxybenzoyl)-benzo[b]furan molecular skeleton, with electron-donating (Me, OMe or OH)
or electron-withdrawing (F, Cl and Br) substituents on the benzene ring, was synthesized and evaluated
for antiproliferative activity, inhibition of tubulin polymerization and cell cycle effects. Adding a methyl
group at the C-3 position resulted in increased activity. The most promising compound in this series was
2-(30,40,50-trimethoxybenzoyl)-3-methyl-6-ethoxy-benzo[b]furan, which inhibits cancer cell growth at
nanomolar concentrations and interacts strongly with tubulin by binding to the colchicine site.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 6 June 2009
Revised 12 August 2009
Accepted 14 August 2009
Available online 20 August 2009
Keywords:
Benzo[b]furan derivatives
Antitubulin agents
Combretastatin-A4
Colchicine
Antiproliferative agents
1. Introduction
rum,4 strongly inhibits the polymerization of tubulin by binding
to the colchicine site.5 Because of its structural simplicity, a wide
The microtubule system of eukaryotic cells plays important
roles in regulating cell architecture, and it has an essential role in
cell division, since microtubules are a key component of the mito-
tic spindle.1 Microtubules are a dynamic cellular compartment in
both neoplastic and normal cells. This dynamicity is characterized
number of CA4 analogues have been developed and evaluated in
SAR studies.6
Among synthetic small molecule tubulin inhibitors, replace-
ment of the olefinic bridge of 1 with a carbonyl group furnished
a benzophenone-type CA4 analogue named phenstatin (2a). This
compound demonstrated interesting efficacy in a variety of tumor
models, while retaining the characteristics of 1.7 The 2-aminoben-
zophenone derivative 2b also strongly inhibited cancer cell growth
and tubulin polymerization and caused mitotic arrest, as did 2a.8
In an earlier publication, we reported a series of methoxy-
substituted 2-(30,40,50-trimethoxybenzoyl)-benzo[b]furan deriva-
tives with general structure 3, with an amino or dimethylamino
substituent at the 3-position of the benzo[b]furan skeleton (com-
pounds 3a and 3b, respectively) as a new class of antimitotic
agents.9a The concomitant presence of a methoxy group at the 6-
position contributed to maximal activity. These compounds, along
with the corresponding benzo[b]thiophene9b,c and indole9d deriva-
tives, inhibited the growth of different cancer cell lines and tubulin
polymerization by binding to the colchicine site of tubulin and
by the continuous turnover of
ab-tubulin heterodimers in the
polymeric microtubules. They are involved in a variety of essential
cellular processes, such as regulation of motility, cell signaling, for-
mation and maintenance of cell shape, as well as transport of
material within the cell.2 Numerous chemically diverse antimitotic
agents, many of which are derived from natural products, have
been found to interact specifically with tubulin.3
Among the microtubule depolymerizing agents, combretastatin
A-4 (CA4, 1; Chart 1) is one of the more studied compounds. CA4,
isolated from the bark of the South African tree Combretum caff-
* Corresponding authors. Tel.: +39 (0)532 455303; fax: +39 (0)532 455953 (R.R.),
tel.: +39 (0)532 455921; fax: +39 (0)532 455953 (P.G.B.).
E-mail addresses: rmr@unife.it (R. Romagnoli), baraldi@unife.it (P.G. Baraldi).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.027

R. Romagnoli et al. / Bioorg. Med. Chem. 17 (2009) 6862–6871
6863
OMe
O
R1
B
MeO
MeO
OMe
OMe
MeO
MeO
R2
R1
A
A
B
R2
OCH3
OMe
O
OH
O
OMe
OMe
3
R1=NH2 or N(Me)2
R2=H, Me or OMe
R1=H, R2=OH, Phenstatin, 2a
R1=NH2, R2=H, 2b
Combretastatin A-4 (CA-4), 1
R1=NH2, R2=6-OCH3, 3a
R1=N(CH3)2, R2=6-OCH3, 3b
OMe
MeO
MeO
OMe
O
R4
R7
R3
OMe
OMe
R5
R6
O
O
4a-x
O
MeO
R3-7=H, 4a
OMe
R4=OMe, R3, 5-7=H, 4b
R5=OMe, R3-4, 6-7=H, 4c
R6=OMe, R3-5, 7=H, 4d
R7=OMe, R3-6=H, 4e
R5=Me, R3-4, 6-7=H, 4f
R5=Cl, R3-4, 6-7=H, 4g
R5=Br, R3-4, 6-7=H, 4h
3c
OMe
OMe
OMe
X
O
MeO
R6=OMe, R7=OH, R3-5=H, 4i
R
5, 6=OMe, R3-4, 7=H, 4j
X=CS, 4y
R5-7=OMe, R3-4=H, 4k
X=CHOH, 4z
X=CHOMe, 4aa
X=CH2, 4ab
R5=OH, R3-4, 6-7=H, 4l
R6=OH, R3-5, 7=H, 4m
R7=OH, R3-6=H, 4n
R3=Me, R4-7=H, 4o
OMe
MeO
R3=Me, R4=OMe, R5-7=H, 4p
R3=Me, R5=OMe, R4, 6-7=H, 4q
R3=Me, R6=OMe, R4-5, 7=H, 4r
R3=Me, R7=OMe, R4-6=H, 4s
R3=Me, R6=OEt, R4-5, 7=H, 4t
R3=Me, R6=F, R4-5, 7=H, 4u
R3=Me, R4=OH, R5-7=H, 4v
R3=Me, R5=OH, R4, 6-7=H, 4w
R3=Me, R6=OH, R4-5, 7=H, 4x
OMe
MeO
O
O
4ac
Chart 1. Inhibitors of tubulin polymerization.
caused G2–M phase arrest of the cell cycle. The higher potency of
the 3-dimethylamino derivative 3b allowed us to verify that an
intramolecular hydrogen bond between the unsubstituted 3-ami-
no group and the carbonyl oxygen of the 2-trimethoxybenzoyl
moiety is not required for activity. These findings prompted us to
study this class of compounds in more detail. We herein describe
the synthesis and structure–activity relationship of 2-(30,40,50-
trimethoxybenzoyl)-benzo[b]furan derivatives in the continuation
of our search for new potent antitubulin agents. We should note
that previous studies have yielded a limited series of tubulin inhib-
itors with the benzo[b]furan molecular skeleton as the core
structure. One of these compounds has structure 3c, which
incorporates the 3-(3,4,5-trimethoxybenzoyl)-6-methoxybenzo[b]
furan ring system.
In a first series (derivatives 4a–x), the 3-amino group of the
benzo[b]furan system was replaced by a hydrogen (4a–n) or a
methyl (4o–x). These molecules possessed either no substituent
(4a and 4o) or a methoxy group at each of the four possible posi-
tions on the benzene ring (compounds 4b–e and 4p–s). In an ef-
fort to design compounds with improved polarity, we decided to
introduce an additional hydroxy group at the C-7 position of com-
pound 4d, to obtain the C-6 methoxy, C-7 hydroxy derivative 4i.
An increase of the polarity in the 2-aroyl benzo[b]furan structure
can be also obtained replacing the methoxy group of compounds
4b–e and 4p–s with a more hydrophilic hydroxyl moiety, to ob-
tain the derivatives 4l–n and 4v–x. The hydroxyl moiety provides
a site for the preparation of a phosphate prodrug, similar to what
has been successfully done in the case of CA4 phosphate.10 Keep-
ing the C-6 methoxy group intact, compounds 4j and 4k were
prepared with the aim of evaluating the effect on biological activ-
ity of one (4j) or two (4k) additional methoxy substituents at the
C-5 and C-5, 7 positions, respectively. Besides the C-5 methoxy
moiety, the substituents examined included electron-donating
methyl (4f) and electron-withdrawing chlorine and bromine (4g
and 4h, respectively) groups. Finally, the 6-methoxy position
(corresponding to the 4-methoxy group in the B-ring of CA4
and 2-aminobenzophenone 2b) of 4r was further studied by syn-
thesizing the corresponding 6-ethoxy (4t) and 6-fluorine (4u)
analogues.
In the present investigation, the 30,40,50-trimethoxyphenyl of the
2-benzoyl moiety was kept unchanged because it is the character-
istic structural requirement for activity in a numerous inhibitors of
tubulin polymerization, such as colchicine, CA4 and podophyllo-
toxin.6 In the current studies we examined the importance of the
3-position of the benzo[b]furan skeleton by studying the effects
of replacing the amino or dimethylamino substituents of com-
pounds with general structure 3 with a hydrogen or a methyl
group, to furnish derivatives with general structure 4. Additional
structure modifications were focused on the C-4/C-7 positions, as
well as on the 2-carbonyl linker of the benzo[b]furan skeleton.

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R. Romagnoli et al. / Bioorg. Med. Chem. 17 (2009) 6862–6871
Starting from compound 4d, additional analogues were gener-
Comparing the 3-unsubstituted derivatives 4a–e and 4l–m with
their 3-methyl congeners 4o–s and 4w–x, the introduction of a
methyl at the C-3 position of the benzo[b]furan system generally
increased antiproliferative activity against all five cell lines. The
antiproliferative activities were also dependent on the substitution
pattern on the benzene part of the benzo[b]furan moiety, with the
most favorable position being C-6 for a methoxy group (4r was
more active than 4p, 4q or 4s, as was observed previously with
the 3-aminobenzo[b]furan derivatives 3a–b), while C-4 was the
best position for an hydroxyl group (4v more active than 4w or
4x). In the absence of the C-3 methyl group, the derivative with
the C-6 methoxyl moiety (4d) was more active than analogues
with the methoxy at other positions (4b, 4c and 4e). Additional
methoxy groups on the benzene ring (4j and 4k) resulted in shar-
ply reduced potency relative to 4d. The introduction of an addi-
tional hydroxyl group at the C-7 position of 4d, to furnish 4i,
caused a substantial loss of activity.
In the series of 3-unsubstituted derivatives, the only compound
with a methoxy moiety with significant activity was 4d and replac-
ing the methoxy with the weaker electron-donating and more
hydrophilic hydroxy group (compound 4m) resulted in a drastic
loss of activity. With the 3-methyl analogues 4p–r, the substitution
of the methoxy group at the C-4 or C-5 positions (4v and 4w) with
the hydroxy function caused an increase in the antiproliferative
activity. A dramatic loss of activity was only observed when the
C-6 methoxy (4r) was replaced with an hydroxy group (4x).
For the inactive C-5 substituted derivative 4c, replacing the
strong electron-donating methoxy function with the electron-
withdrawing chlorine (4g) or bromine (4h) groups had no effect
on activity, but replacement of the C-5 methoxy group with the
moderately electron-donating methyl moiety (4f) slightly in-
creased activity.
ated by modification of the 2-carbonyl function, which was con-
verted into thiocarbonyl (4y), reduced to carbinol (4z) and to
methylene (4ab) or transformed into a methyl ether (4aa).
Finally, by the synthesis of tetracyclic compound 4ac, the phe-
nyl of the 2-(30,40,50-trimethoxy)benzoyl moiety and the 3-position
of the benzo[b]furan skeleton were locked in a conformation in
which these two ring were forced to be coplanar.
We should note that previous studies have yielded a limited
series of tubulin inhibitors with the benzo[b]furan molecular skel-
eton as the core structure. These compounds have general struc-
ture 6, which incorporates the 3-(3,4,5-trimethoxybenzoyl)-6-
methoxybenzo[b]furan ring system.11
2. Chemistry
The 2-(30,40,50-trimethoxybenzoyl)benzo[b]furan derivatives
4a–l, 4n–w, 6m and 6x were synthesized by a ‘one-step’ condensa-
tion of the corresponding, variously substituted salicylaldehydes
5a–n or 2-hydroxyacetophenones 5o–x with 2-bromo-1-(3,4,5-tri-
methoxyphenyl)ethanone9b and anhydrous potassium carbonate
in refluxing acetone (Scheme 1). For the tert-butyldimethylsilyl
(TBDMS) analogues 5l, 5n and 5v–w, the cyclization was accom-
plished by the concomitant removal of TBDMS-protecting group,
to afford derivatives 4l, 4n and 4v–w. The 6-hydroxy-2-aroyl ben-
zo[b]furan derivatives 4m and 4x were obtained by cleavage of the
6-benzyloxy group of 6m and 6x, respectively, with a mixture of
activated palladium on charcoal and ammonium formate.
The treatment of the 2-carbonyl moiety of 4d with Lawesson0s
reagent or its reduction with sodium borohydride furnished the
thiocarbonyl or the carbinol derivatives 4y and 4z, respectively.12
This latter compound was further converted to the methyl ether
4aa by treatment with pyridinium p-toluensulfonate in a mixture
of MeOH–THF or reduced to the methylene derivative 4ab with tri-
ethylsilane in trifluoroacetic acid. The cyclic ketone 4ac was syn-
thesized by intramolecular cross-coupling of bromo derivative 7
catalyzed with palladium (0) tetrakistriphenylphosphine in N,N-
dimethylacetamide at 130 °C. Derivative 7 was obtained by the
chemoselective bromination of 4d with NBS in acetonitrile.13
As noted above, the activity of 4r was increased 10-fold against
L1210, FM3A, Molt/4 and CEM cells and 2-fold for HeLa cells when
the C-6 methoxy group was replaced with an ethoxy moiety (4t). In
contrast, an electron-withdrawing fluorine group at C-6 (4u)
caused a dramatic reduction of antiproliferative activity.
Linking the ortho-position of the phenyl of the 2-(30,40,50-trim-
ethoxybenzoyl)moiety, to the 3-position of benzo[b]furan moiety,
to yield the conformationally constrained derivative 4ac, led to a
completely inactive compound.
To confirm that the antiproliferative activities of these com-
pounds were related to an interaction with the microtubule sys-
tem, the most active compounds, 4b, 4d, 4p–r, 4t, 4v and 4w,
along with the 6-fluoro derivative 4u, were evaluated for their
in vitro inhibition of tubulin polymerization and for their inhibi-
tory effects on the binding of [3H]colchicine to tubulin (in the latter
3. Biological results and discussion
Table 1 summarizes the growth inhibitory effects of 2-(30,40,50-
trimethoxy)benzo[b]furan derivatives 4a–ac against murine leuke-
mia (L1210), murine mammary carcinoma (FM3A), human T-lym-
phoblastoid (Molt/4 and CEM) and human cervix carcinoma (HeLa)
cells, with CA4 (1), 3a and 3b as reference compounds. The 3-
methyl-6-ethoxy derivative 4t possessed the highest potency,
inhibiting the growth of L1210, FM3A, Molt/4, CEM and HeLa cells
with IC50 values of 2.0, 2.8, 1.2, 2.8 and 6.3 nM, respectively. These
values are similar to those obtained with CA4 and from 20- to 100-
fold higher activity than those obtained with the 3-amino deriva-
tives 3a and 3b. However, this marked improvement in activity rel-
ative to 3a–b may derive in part from the ethoxy substituent at C-
6, since the analogue with a methoxyl at C-6 (4r) was approxi-
mately 10-fold less active than 4t in four of the cell lines.
Comparing the 6-methoxy derivatives 3a–b, 4d and 4r, the order
of activity for the substituent at the 3-position of the benzo[b]furan
moiety was methyl (4r) > hydrogen (4d) ꢀ dimethylamino (3b) >
amino (3a). With the four compounds IC50 values ranged from 3 to
27 nM for 4r, from 59 to 110 nM for 4d, from 48 to 78 for 3b, and
from 87 to 430 nM for 3a. Comparing the activities of the carbonyl
derivative 4d with those of derivatives 4y–z and 4aa–ab indicated
that the 2-carbonyl moiety could only be replaced with a thiocar-
bonyl group (4y) among the bridging moieties examined.
assay, tubulin was examined at a concentration of 1
lM, while
compounds and colchicine were at 5
l
M).14,15 For comparison,
CA4 was examined in contemporaneous experiments as a refer-
ence compound (Table 2). The benzo[b]furan derivatives 4d, 4r,
4t and 4v with IC50 values of 0.70, 0.55, 0.43 and 0.57
tively, exhibited antitubulin activity about two times greater than
that of CA4 (1.0 M), while 4b, 4q and 4w had IC50 values of 1.1–
1.2 M, essentially equivalent to that of CA4.
The order of inhibitory action on tubulin assembly was
lM, respec-
l
l
4t > 4r > 4v > 4d > CA4 > 4b > 4w > 4q > 4p >> 4u, which was con-
sistent with the results of the antiproliferative assays, except that
4d was more potent than 4v and 4p was more potent than 4q.
The most potent compound in this series was compound 4t, with
an IC50 value of 0.43 lM, which correlates well with its having
the greatest antiproliferative activity. Compounds 4b, 4q and 4w
were as active as CA4 as inhibitors of tubulin assembly, although
both compounds were less active in their effects on cell growth.
Compound 4u showed weak antitubulin polymerization activity,
which is consistent with its low antiproliferative activity.

R. Romagnoli et al. / Bioorg. Med. Chem. 17 (2009) 6862–6871
6865
OMe
R3
OMe
R1=H, 4m
R1=Me, 4x
OMe
O
HO
O
c
OMe
R4
R7
R3
OMe
OMe
R5
R6
OMe
R4
O
O
OMe
OMe
R5
R6
O
a
b
R3
OH
4a-l and 4n-w, 6m and 6x
O
MeO
6m, R3=H, R6=OBn
S
R7
5a-x
4y
6x, R3=Me, R6=OBn
d
R3-7=H, 5a
g
R4=OMe, R3, 5-7=H, 5b
R5=OMe, R3-4, 6-7=H, 5c
R6=OMe, R3-5, 7=H, 5d
R7=OMe, R3-6=H, 5e
R5=Me, R3-4, 6-7=H, 5f
R5=Cl, R3-4, 6-7=H, 5g
R5=Br, R3-4, 6-7=H, 5h
OMe
OMe
OMe
OMe
Br
OMe
OMe
O
O
7
MeO
MeO
MeO
4z
OH
O
R6=OMe, R7=OH, R3-5=H, 5i
R5, 6=OMe, R3-4, 7=H, 5j
R
5-7=OMe, R3-4=H, 5k
e or f
h
R5=OTBDMS, R3-4, 6-7=H, 5l*
R6=OBn, R3-5, 7=H, 5m
OMe
OMe
O
R7=OTBDMS, R3-6=H, 5n*
R3=Me, R4-7=H, 5o
MeO
OMe
OMe
OMe
R3=Me, R4=OMe, R5-7=H, 5p
R3=Me, R5=OMe, R4, 6-7=H, 5q
R3=Me, R6=OMe, R4-5, 7=H, 5r
R3=Me, R7=OMe, R4-6=H, 5s
R3=Me, R6=OEt, R4-5, 7=H, 5t
R3=Me, R6=F, R4-5, 7=H, 5u
O
MeO
R2
O
R2=OMe, 4aa
R2=H, 4ab
4ac
R3=Me, R4=OTBDMS, R5-7=H, 5v*
R3=Me, R5=OTBDMS, R4, 6-7=H, 5w*
R3=Me, R6=OBn, R4-5, 7=H, 5x
Scheme 1. Reagents and conditions: (a) (3,4,5-Trimethoxyphenyl)-2-bromo-ethanone, K2CO3, (CH3)2CO, rt; (b) Lawesson’s reagent, THF, rt from 4d; (c) HCO2NH4, 10% Pd/C,
MeOH, rt from 6m and 6x; (d) NaBH4, MeOH, rt from 4d; (e) PTSA, MeOH–THF, rt; (f) Et3SiH, TFA, CH2Cl2, rt; (g) NBS, benzoyl peroxide, MeCN, rt from 4d; (h) Pd(Ph3P)4, KOAc,
*
DMA, 130 °C. For compounds 5l, 5n and 5v–w, condition ‘a’ led to a cyclization with concomitant removal of TBDMS group, to afford 4l, 4n and 4v–w, respectively.
In the colchicine binding studies, compounds 4r and 4t strongly
inhibited the binding of [3H]colchicine to tubulin, since 97% and
96% inhibition, respectively, occurred with these agents and colchi-
Molecular docking studies on this series of compounds in the
colchicine site of tubulin were also performed. There was a compa-
rable binding mode of these compounds to that of DAMA-colchi-
cine co-crystallized in the tubulin structure used,16 with the
trimethoxyphenyl moiety occupying the same pocket as the corre-
sponding ring A of the colchicinoid (Fig. 2).
cine, both at 5 lM. These derivatives were as active as CA4, which
in these experiments inhibited colchicine binding by 99%. These
data indicate that 4r and 4t strongly bind to the colchicine site
on tubulin.
Thesubstituentintheposition6 ofthebenzofuran analogues, like
the methoxy group on ring C of colchicinoids, falls into a hydropho-
bic region deep in the binding site (shown in green inFig. 3). Further-
more, a polar area on the tubulin surface corresponding to the
carbonylgroup of Thr179(residue numbering derived from the crys-
tal structure used) with potential for accepting a hydrogen bond is
closetotheC-4positionof thebenzo[b]furangroup(showninpurple
inFig. 3). This feature could be exploited by the hydroxyl group of 4v
to establish a hydrogen bond with tubulin, which could compensate
to the loss of the hydrophobic contact provided by the C-6 methoxy
group, which is missing in 4v, and justify the activity of this com-
pound in the tubulin polymerization assay. It should be mentioned
that the position observed for the hydroxyl group of 4v is similar
to the proposed binding location of the corresponding functional
group of CA4, although in the case of 4v is the carbonyl group of
Thr179 that is closer to the inhibitor (2.54 Å), while for CA4 the hy-
droxyl group is placed close to Val181 (see Fig. 1, Supplementary
data).17
Because molecules exhibiting effects on tubulin assembly
should cause alteration of cell cycle parameters with preferential
G2–M blockade, flow cytometry analysis was performed to deter-
mine the effect of the compounds 4b, 4d, 4p–r, 4t and 4u–w on
K562 (human chronic myelogenous leukemia) cells. Cells were cul-
tured for 24 h in the presence of each compound at the concentra-
tion able to inhibit 100% cell growth after 24 h (4b = 980 nM, 4d
and 4p = 420 nM, 4q = 230 nM, 4r = 26 nM, 4t = 16 nM, 4u = 1.62
lM, 4v = 530 nM, 4w = 480 nM). Analysis of sub-G0–G1 (apoptotic
peak, A), G0–G1, S, and G2–M peaks revealed that the compounds
caused somewhat different effects on cell cycle distribution
(Fig. 1). All tested compounds caused an increase in the proportion
of cells in the G2–M peak relative to the untreated control, and
compounds 4p and 4u also caused cells to accumulate in late S
phase. For compounds 4b, 4p. 4u and 4w, the accumulation of cells
in G2–M phase was accompanied by the appearance of a significant
sub-G0–G1 peak (A = 20–44%) due to apoptosis.

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R. Romagnoli et al. / Bioorg. Med. Chem. 17 (2009) 6862–6871
Table 1
tubulin leads to cell cycle arrest in the G2–M phase and to an apop-
totic cell death. Our structure–activity relationship study indicates
that a methoxy group located at the C-6 position of the benzo[b]-
furan ring yields the most active compound. Changing its position
from C-6 to C-4, C-5 or C-7 led to a reduction in potency. Replace-
ment of methoxy by the more hydrophilic hydroxyl group turned
out to be detrimental to potency in the case of the 2-unsubstituted
benzo[b]furan series. The 2-(30,40,50-trimethoxybenzoyl)-3-methyl-
6-ethoxybenzo[b]furan derivative 4t was the most potent ana-
logue, with IC50 values ranging from 1.2 to 6.3 nM, in the same
range as the values obtained with CA4. Compound 4t also had
excellent potency as an inhibitor of tubulin polymerization
In vitro inhibitory effects of compounds 3a–b, 4a–ac and CA4 against the proliferation
of murine leukemia (L1210), murine mammary carcinoma (FM3A), human T-
lymphocyte (Molt/4 and CEM) and human cervix carcinoma (HeLa) cells
Compound
IC50 (nM)a
Molt4/C8
L1210
FM3A/0
CEM/0
Hela
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
>10,000
410 23
>10,000
>10,000
550 42
>10,000
100 10
>10,000
8000 100 >10,000
360 10 870 50
9400 500 >10,000
59 12 70 21
7800 290 >10,000
7900 100
410 16
>10,000
110 20
>10,000
90
2
>10,000
7100 580 8400 300 2500 160 7200 210 >10
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
(IC50 = 0.43 lM). At the 2-position of the benzo[b]furan, the linker
is much more effective as a carbonyl than as carbinol, methoxy-
methyl or simple methylene group. A thiocarbonyl linker, however,
was compatible with good activity.
1800 400 1200 400 770 20
1900 400 570 110
>10,000
>10,000
>10,000
>10,000
2100 200 2100 10
590 40 570 30
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1300 80
200 70
>10,000
>10,000
>10,000
>10,000
2000 50
490 10
>10,000
>10,000
>10,000
>10,000
890 130
340 30
1900 200
3.2 0.6
5. Experimental
5.1. Chemistry
2300 400 4400 390 1900 100 400 150
24 27 19 20
1700 400 1700 100 400 30
2.0 1.2 2.8 1.0 1.2 0.8
3500 200 3700 100 2100
0
7
2
2
4s
4t
2400 150 530 28
2.8 0.6 6.3 2.7
2700 200 2000
5.1.1. Materials and methods
4u
4v
4w
4x
4y
4z
4aa
4ab
4ac
3a
3b
CA4
0
0
2-Hydroxybenzaldehyde (5a), 2-hydroxy-6-methoxybenzalde-
hyde (5b), 2-hydroxy-5-methoxybenzaldehyde (5c), 2-hydroxy-4-
methoxybenzaldehyde (5d), 2-hydroxy-4-methoxybenzaldehyde
(5e), 2-5-hydroxy-5-methylbenzaldehyde (5f), 2-hydroxy-5-chloro-
benzaldehyde (5g), 2-hydroxy-5-bromobenzaldehyde (5h), 2-hydro-
480 32
800 31
>10,000
330 10
430 27
>10,000
320 40
84 21
100 10
430
130 24
290 10
240 30
1300 90
100 10
0
4300 300 260 16
220 80
180
0
100 25
>10,000
4400 290
>10,000
>10,000
7009 530 3005 120 >10,000
>10,000 >10,000 >10,000
2300 400 2600 600 770 43
xy-3-methoxybenzaldehyde
(5i),
2-hydroxy-4-(benzyloxy)
1500 300 680 10
>10,000
87 22
benzaldehyde (5m), 1-(2-hydroxyphenyl)ethanone (5o), 2-hydroxy-
6-methoxyacetophenone (5p), 2-hydroxy-5-methoxyacetophenone
(5q), 2-hydroxy-4-methoxyacetophenone (5r), 2-hydroxy-4-ethoxy-
acetophenone (5t), 2-hydroxy-4-fluoroacetophenone (5u) are com-
mercially available and were used as received. For the preparation of
5j–l, 5n, 5s, 5v–x see Ref. 18.
>10,000
430 40
>10,000
280 160
>10,000
140 20
>10,000
n.d.
n.d.
65
5
59
5
48
4
78
3
2.8 1.1
42 6.0
16 1.4
1.9 1.6
1.9 1.6
n.d. = not determined.
a
IC50 = Compound concentration required to inhibit tumor cell proliferation by
50%. Data are expressed as the mean SE from the dose–response curves of at least
three independent experiments.
1H NMR spectra were recorded on a Bruker AC 200 spectrometer.
Chemical shifts (d) are given in ppm upfield from tetramethylsilane
as internal standard, and the spectra were recorded in appropriate
deuterated solvents, as indicated. Melting points (mp) were deter-
minedon a Buchi-Tottoli apparatus andareuncorrected. Allproducts
reported showed 1H NMR spectra in agreement with the assigned
structures. Elemental analyses were conducted by the Microanalyti-
calLaboratory of the Chemistry Departmentof the University of Ferr-
ara. Mass spectra were obtained by electrospray ionisation (ESI) in
positive mode using a ESI Micromass ZMD 2000 mass spectrometer.
Allreactions were carried outunder an inertatmosphere of drynitro-
gen, unless otherwise described. Standard syringe techniques were
applied for transferring dry solvents. Reaction courses and product
mixtures were routinely monitored by TLC on silica gel (precoated
F254 Merck plates) and visualized with aqueous KMnO4. Flash chro-
matography was performed using 230–400 mesh silica gel and the
indicated solvent system. Organic solutions were dried over anhy-
drous Na2SO4. Calcium chloride was used in the distillation of DMF,
and the distilled solvent was stored over molecular sieves (3 Å).
Table 2
Inhibition of tubulin polymerization and colchicine binding by compounds 4b, 4d,
4p–r, 4t–w and CA4
Compound Tubulin assemblya IC50 SD (
l
M) Colchicine bindingb
%
SD
4b
4d
4p
4q
4r
4t
4u
4v
1.1 0.1
0.70 0.08
1.8 0.2
76
79
66
65
97
96
n.d.
85
69
99
2
0
0
0
1
1
1.2 0.1
0.55 0.03
0.43 0.06
6.9 0.8
0.57 0.03
1.1 0.1
1
3
2
4w
CA4 (1)
1.0 0.1
n.d.: not determined.
a
Inhibition of tubulin polymerization. Tubulin was at 10
l
M.
Inhibition of [3H]colchicine binding. Tubulin, colchicine and tested compound
were at 1, 5 and 5 M, respectively.
b
l
5.2. General procedure for the synthesis of 2-(30,40,50-
trimethoxybenzoyl)-3-amino benzofuranes (4a–l, 4n–w, 6m
and 6x)
4. Conclusions
The SAR information indicated that the introduction of methyl
at the C-3 position of the benzo[b]furan moiety resulted in in-
creased activity compared with the corresponding 3-amino coun-
terpart,9a thus revealing that this latter substituent is not
essential for activity. Several compounds (4d, 4r, 4t and 4v)
showed excellent activity as inhibitors of tubulin polymerization,
and were more potent than CA4 in this assay. The interaction with
To a solution of the appropriate substituted salicylaldehyde 5a–
or 2-hydroxyacetophenone 5o–x (1 mmol) in dry acetone
(15 mL) was added 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone
(289 mg, 1 mmol) and anhydrous potassium carbonate (276 mg,
2 mmol) while stirring, and the reaction mixture was refluxed for
18 h. After cooling, the reaction mixture was evaporated, and the
n

R. Romagnoli et al. / Bioorg. Med. Chem. 17 (2009) 6862–6871
6867
Figure 1. Effects of compounds 4d (a), 4b (b), 4q (c), 4r (d), 4p (e), 4w (f), 4v (g), 4t (h) and 4u (i) on DNA content/cell following the treatment of K562 cells for 24 h. Cell cycle
distribution was analyzed by the standard propidium iodide procedure as described in Section 5. Control is reported in Figure 1 as panel j.
residue was dissolved in a mixture of dichloromethane (15 mL)
and water (5 mL). The organic layer was washed with brine, dried
and concentrated under reduced pressure to obtain a residue,
which was purified by flash column chromatography. The final
product was recrystallized from petroleum ether.
1H NMR (CDCl3) d: 3.95 (s, 3H), 3.96 (s, 6H), 3.98 (s, 3H), 7.12 (m,
2H), 7.34 (s, 2H), 7.49 (s, 1H), 7.52 (d, J = 9.2 Hz, 1H). Anal. Calcd
for C19H18O6: C, 66.66; H, 5.30. Found: C, 66.47; H, 5.12.
5.2.3. (5-Methoxybenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4c)
5.2.1. (Benzofuran-2-yl)(3,4,5-trimethoxyphenyl)methanone
(4a)
The residue was chromatographed with EtOAc/petroleum ether
2:8 as eluent to give 4c as a white solid, yield: 83%, mp 154–156 °C.
1H NMR (CDCl3) d: 3.88 (s, 3H), 3.96 (s, 6H), 3.97 (s, 3H), 6.70 (d,
J = 7.6 Hz, 1H), 7.25 (m, 1H), 7.32 (s, 2H), 7.43 (d, J = 8.4 Hz, 1H),
7.62 (d, J = 1.0 Hz, 1H). Anal. Calcd for C19H18O6: C, 66.66; H,
5.30. Found: C, 66.38; H, 5.03.
The residue was chromatographed with EtOAc/petroleum ether
3:7 as eluent to give 4a as a white solid, yield: 93%, mp 94–96 °C.
1H NMR (CDCl3) d: 3.95 (s, 6H), 3.96 (s, 3H), 7.36 (m, 3H), 7.50 (t,
J = 8.0 Hz, 1H), 7.53 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.76 (d,
J = 8.0 Hz, 1H). Anal. Calcd for C18H16O5: C, 69.22; H, 5.16. Found:
C, 69.01; H, 5.02.
5.2.4. (6-Methoxybenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4d)
5.2.2. (4-Methoxybenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4b)
The residue was chromatographed with EtOAc/petroleum ether
3:7 as eluent to give 4b as a white solid, yield: 85%, mp 118–120 °C.
The residue was chromatographed with EtOAc/petroleum ether
3:7 as eluent to give 4d as a white solid, yield: 89%, mp 115–
117 °C. 1H NMR (CDCl3) d: 3.99 (s, 3H), 3.94 (s, 6H), 3.95 (s, 3H),
6.97 (dd, J = 8.8 and 2.2 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H), 7.29 (s,

6868
R. Romagnoli et al. / Bioorg. Med. Chem. 17 (2009) 6862–6871
for C18H15ClO5: 62.35; H, 4.36; Cl, 10.22. Found: 62.06; H, 4.14;
Cl, 10.01.
5.2.8. (5-Bromobenzofuran-2-yl)(3,4,5-trimethoxyphenyl)meth-
anone (4h)
The residue was chromatographed with EtOAc/petroleum ether
2:8 as eluent to give 4h as a white solid, yield: 83%, mp 144–
146 °C. 1H NMR (CDCl3) d: 3.94 (s, 6H), 3.96 (s, 3H), 7.33 (s, 2H),
7.47 (s, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.56 (dd, J = 8.8 and 2.0 Hz,
1H), 7.88 (d, J = 2.0 Hz, 1H). Anal. Calcd for C18H15BrO5: C, 55.26;
H, 3.86; Br, 20.42. Found: C, 55.02; H, 3.68; Br, 20.17.
5.2.9. (7-Hydroxy-6-methoxybenzofuran-2-yl)(3,4,5-trime-
thoxyphenyl)methanone (4i)
The residue was chromatographed with EtOAc/petroleum ether
4:6 as eluent to give 4i as a white solid, yield: 83%, mp 128–130 °C.
1H NMR (CDCl3) d: 3.91 (s, 3H), 3.93 (s, 6H), 3.95 (s, 3H), 6.97 (d,
J = 8.6 Hz, 1H), 7.28 (s, 2H), 7.32 (d, J = 8.6 Hz, 1H), 7.50 (s, 1H),
10.2 (s, 1H). Anal. Calcd for C19H18O7: C, 63.68; H, 5.06. Found: C,
63.50; H, 4.88.
Figure 2. Putative binding mode of compound 4r (colored in purple); DAMA-
colchicine colored in green.
5.2.10. (5,6-Dimethoxybenzofuran-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (4j)
The residue was chromatographed with EtOAc/petroleum ether
4:6 as eluent to give 4j as a yellow solid, yield: 87%, mp 145–
147 °C. 1H NMR (CDCl3) d: 3.94 (s, 6H), 3.95 (s, 6H), 3.98 (s, 3H),
7.08 (s, 1H), 7.11 (s, 1H), 7.29 (s, 2H), 7.47 (s, 1H). Anal. Calcd for
C20H20O7: C, 64.51; H, 5.41. Found: C, 64.40; H, 5.20.
5.2.11. (5,6,7-Trimethoxybenzofuran-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (4k)
The residue was chromatographed with EtOAc/petroleum ether
4:6 as eluent to give 4k as a yellow solid, yield: 73%, mp 100–102 °C.
1H NMR (CDCl3) d: 3.92 (s, 3H), 3.93 (s, 3H), 3.95 (s, 3H), 3.96 (s, 3H),
3.97 (s, 3H), 4.24 (s, 3H), 6.84 (s, 1H), 7.40 (s, 2H), 7.51 (s, 1H). Anal.
Calcd for C21H22O8: C, 62.68; H, 5.51. Found: C, 62.44; H, 5.38.
Figure 3. Putative binding mode of compound 4r (colored in gray) and 4v (colored
in yellow). Lipophilic potential map represented in green; H-bond donor potential
map represented in purple.
5.2.12. (5-Hydroxybenzofuran-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (4l)
The residue was chromatographed with EtOAc/petroleum ether
4:6 as eluent to give 4l as a yellow solid, yield: 80%, mp 155–
157 °C. 1H NMR (CDCl3) d: 3.94 (s, 3H), 3.96 (s, 6H), 7.06 (dd,
J = 8.8 and 2.4 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 7.33 (s, 2H), 7.44
(s, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.52 (s, 1H). Anal. Calcd for
C18H16O6: C, 65.85; H, 4.91. Found: C, 65.68; H, 4.67.
2H), 7.48 (s, 1H), 7.60 (d, J = 8.8 Hz, 1H). Anal. Calcd for C19H18O6: C,
66.66; H, 5.30. Found: C, 66.42; H, 5.14.
5.2.5. (7-Methoxybenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4e)
The residue was chromatographed with EtOAc/petroleum ether
3:7 as eluent to give 4e as a yellow solid, yield: 95%, mp 116–
118 °C. 1H NMR (CDCl3) d: 3.95 (s, 3H), 3.96 (s, 6H), 4.03 (s, 3H),
6.96 (d, J = 7.6 and 2.0 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.33 (d,
J = 7.6 Hz, 1H), 7.44 (s, 2H), 7.57 (s, 1H). Anal. Calcd for C19H18O6:
C, 66.66; H, 5.30. Found: C, 66.39; H, 5.09.
5.2.13. (7-Hydroxybenzofuran-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (4n)
The residue was chromatographed with EtOAc/petroleum ether
3:7 as eluent to give 4n as a yellow solid, yield: 74%, mp 166–
168 °C. 1H NMR (CDCl3) d: 3.94 (s, 3H), 3.96 (s, 6H), 7.07 (d, J = 7.6
and 2.0 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.28 (m, 3H), 7.53 (s, 2H).
Anal. Calcd for C18H16O6: C, 65.85; H, 4.91. Found: C, 65.59; H, 4.39.
5.2.6. (3,4,5-Trimethoxyphenyl)(5-methylbenzofuran-2-
yl)methanone (4f)
The residue was chromatographed with EtOAc/petroleum ether
3:7 as eluent to give 4f as a white solid, yield: 88%, mp 106–107 °C.
1H NMR (CDCl3) d: 2.47 (s, 3H), 3.94 (s, 6H), 3.96 (s, 3H), 7.30 (d,
J = 9.2 Hz, 1H), 7.33 (s, 2H), 7.47 (s, 1H), 7.52 (m, 2H). Anal. Calcd
for C19H18O5: C, 69.93; H, 5.56. Found: C, 69.77; H, 5.33.
5.2.14. (3,4,5-Trimethoxyphenyl)(3-methylbenzofuran-2-yl)-
methanone (4o)
The residue was chromatographed with EtOAc/petroleum ether 1:9
aseluent to give 40 asawhitesolid, yield:85%, mp108–110 °C. 1HNMR
(CDCl3) d: 2.65 (s, 3H), 3.94 (s, 6H), 3.96 (s, 3H), 7.36 (t, J = 7.2 Hz, 1H),
7.50 (s, 2H), 7.52 (m, 2H), 7.70 (dd, J = 8.0 and 1.0 Hz, 1H). Anal. Calcd
for C19H18O5: C, 69.93; H, 5.56. Found: C, 69.68; H, 5.38.
5.2.7. (5-Chlorobenzofuran-2-yl)(3,4,5-trimethoxyphenyl)meth-
anone (4g)
The residue was chromatographed with EtOAc/petroleum ether
2:8 as eluent to give 4g as a white solid, yield: 72%, mp 136–138 °C.
1H NMR (CDCl3) d: 3.95 (s, 6H), 3.97 (s, 3H), 7.33 (s, 2H), 7.44 (m,
2H), 7.56 (d, J = 8.8 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H). Anal. Calcd
5.2.15. (4-Methoxy-3-methylbenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4p)
The residue was chromatographed with EtOAc/petroleum
ether 2:8 as eluent to give 4p as a white solid, yield: 90%, mp

R. Romagnoli et al. / Bioorg. Med. Chem. 17 (2009) 6862–6871
6869
107–109 °C. 1H NMR (CDCl3) d: 2.78 (s, 3H), 3.9 (s, 3H), 3.95 (s,
6H), 3.96 (s, 3H), 6.65 (d, J = 7.6 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H),
7.37 (m, 3H). Anal. Calcd for C20H20O6: C, 67.41; H, 5.66. Found:
C, 67.20; H, 5.38.
5.2.23. (6-(Benzyloxy)benzofuran-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (6m)
The residue was chromatographed with EtOAc/petroleum ether
4:6 as eluent to give 6m as a pink solid, yield: 61%, mp 182–184 °C.
1H NMR (CDCl3) d: 3.94 (s, 3H), 3.96 (s, 6H), 4.35 (s, 2H), 6.51 (d,
J = 2.4 Hz, 1H), 7.10 (dd, J = 8.6 and 2.4 Hz, 1H), 7.28 (m, 5H), 7.29
(s, 2H), 7.47 (s, 1H), 7.61 (d, J = 8.6 Hz, 1H). ESI-MS m/z 419.1
[M+1]+.
5.2.16. (5-Methoxy-3-methylbenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4q)
The residue was chromatographed with EtOAc/petroleum ether
2:8 as eluent to give 4q as a white solid, yield: 81%, mp 122–124 °C.
1H NMR (CDCl3) d: 2.62 (s, 3H), 3.90 (s, 3H), 3.93 (s, 6H), 3.96 (s,
3H), 7.07 (m, 1H), 7.12 (d, J = 8.6 Hz, 1H), 7.42 (m, 3H). Anal. Calcd
for C20H20O6: C, 67.41; H, 5.66. Found: C, 67.12; H, 5.24.
5.2.24. (6-(Benzyloxy)-3-methylbenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (6x)
The residue was chromatographed with EtOAc/petroleum ether
2:8 as eluent to give 6x as a white solid, yield: 68%, mp 110–112 °C.
1H NMR (CDCl3) d: 2.61 (s, 3H), 3.93 (s, 3H), 3.95 (s, 6H), 5.15 (s,
2H), 7.07 (m, 2H), 7.37 (s, 2H), 7.44 (m, 5H), 7.56 (d, J = 8.6 Hz,
1H). ESI-MS m/z 433.2 [M+1]+.
5.2.17. (6-Methoxy-3-methylbenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4r)
The residue was chromatographed with EtOAc/petroleum
ether 2:8 as eluent to give 4r as a white solid, yield: 77%, mp
101–103 °C. 1H NMR (CDCl3) d: 2.61 (s, 3H), 3.89 (s, 3H), 3.94
(s, 6H), 3.96 (s, 3H), 6.99 (m, 2H), 7.39 (s, 2H), 7.59 (d,
J = 9.6 Hz, 1H). Anal. Calcd for C20H20O6: C, 67.41; H, 5.66. Found:
C, 67.19; H, 5.44.
5.3. General procedure for the synthesis of 4m and 4x
A suspension of benzyloxy derivative 6m or 6x (1 mmol)
ammonium formate (630 mg, 10 mmol) and activated Pd on char-
coal (10%, 150 mg) in a mixture of THF/MeOH (1:1, 20 mL) was
heated to reflux for 1 h. The catalyst was filtered over Celite, the
solvent removed under reduced pressure and the product purified
by column chromatography (silica gel, EtOAc/petroleum ether 1:1).
5.2.18. (7-Methoxy-3-methylbenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4s)
The residue was chromatographed with EtOAc/petroleum
ether 2:8 as eluent to give 4s as a white solid, yield: 71%, mp
143–145 °C. 1H NMR (CDCl3) d: 2.66 (s, 3H), 3.95 (s, 3H), 3.97
(s, 6H), 4.00 (s, 3H), 6.97 (dd, J = 6.0 and 2.8 Hz, 1H), 7.27 (m,
2H), 7.53 (s, 2H). Anal. Calcd for C20H20O6: C, 67.41; H, 5.66.
Found: C, 67.27; H, 5.49.
5.3.1. (6-Hydroxybenzofuran-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (4m)
The residue was chromatographed with EtOAc/petroleum ether
1:1 as eluent to give 4m as a yellow solid, yield: 46%, mp 141–
143 °C. 1H NMR (CDCl3) d: 3.84 (s, 3H), 3.88 (s, 6H), 6.87 (dd,
J = 8.6 and 2.0 Hz, 1H), 6.92 (s, 1H), 7.23 (s, 2H), 7.66 (d,
J = 8.4 Hz, 1H), 7.77 (s, 1H), 10.1 (s, 1H). Anal. Calcd for C18H16O6:
C, 65.85; H, 4.91. Found: C, 65.58; H, 4.72.
5.2.19. (6-Ethoxy-3-methylbenzofuran-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (4t)
The residue was chromatographed with EtOAc/petroleum ether
1.5:8.5 as eluent to give 4t as a yellow solid, yield: 81%, mp 98–
100 °C. 1H NMR (CDCl3) d: 1.41 (t, J = 7.2 Hz, 3H), 2.60 (s, 3H),
3.93 (s, 6H), 3.95 (s, 3H), 4.12 (q, J = 7.2 Hz, 2H), 6.97 (m, 2H),
7.38 (s, 2H), 7.55 (d, J = 9.2 Hz, 1H). Anal. Calcd for C21H22O6: C,
68.10; H, 5.99. Found: C, 67.89; H, 5.79.
5.3.2. (6-Hydroxy-3-methylbenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4x)
The residue was chromatographed with EtOAc/petroleum ether
1:1 as eluent to give 4x as a yellow solid, yield: 46%, mp 211–
213 °C. 1H NMR (CDCl3) d: 2.08 (s, 3H), 3.77 (s, 3H), 3.85 (s, 6H),
6.86 (dd, J = 8.6 and 2.0 Hz, 1H), 6.99 (d, J = 2.0 Hz, 1H), 7.29 (s,
2H), 7.62 (d, J = 8.6 Hz, 1H), 10.1 (bs, 1H). Anal. Calcd for
C19H18O6: C, 66.66; H, 5.30. Found: C, 66.38; H, 5.08.
5.2.20. (6-Fluoro-3-methylbenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4u)
The residue was chromatographed with EtOAc/petroleum ether
1:9 as eluent to give 4u as a brown solid, yield: 79%, mp 130–
132 °C. 1H NMR (CDCl3) d: 2.62 (s, 3H), 3.93 (s, 6H), 3.96 (s, 3H),
7.08 (dd, J = 9.0 and 2.6 Hz, 1H),), 7.16 (dd, J = 9.4 and 2.2 Hz,
1H), 7.38 (s, 2H), 7.66 (dd, J = 8.8 and 5.2 Hz, 1H). Anal. Calcd for
C19H17FO5: C, 66.27; H, 4.98. Found: C, 66.03; H, 4.76.
5.3.3. Synthesis of (6-methoxybenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanethione (4y)
To a magnetically stirred solution of 4d (1 mmol) in anhydrous
THF at room temperature was added Lawesson0s reagent
(1.1 mmol), and the mixture was stirred for 1 h. The solvent was
evaporated in vacuo, and the residue was dissolved in dichloro-
methane (15 mL). The organic extract was washed with water
(5 mL), dried over Na2SO4 and concentrated in vacuo. The residue
purified by flash chromatography (ethyl acetate/petroleum ether
3:7) furnished 4y as a white powder, yield: 96%, mp 143–145 °C.
1H NMR (CDCl3) d: 3.90 (s, 3H), 3.92 (s, 6H), 3.94 (s, 3H), 6.94
(dd, J = 8.8 and 2.4 Hz, 1H), 7.10 (s, 2H), 7.12 (d, J = 2.4 Hz, 1H),
7.37 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H). Anal. Calcd for C19H18O5S: C,
63.67; H, 5.06; S, 8.95. Found: C, 63.48; H, 4.88; S, 8.74.
5.2.21. (4-Hydroxy-3-methylbenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4v)
The residue was chromatographed with EtOAc/petroleum ether
3:7 as eluent to give 4v as a yellow solid, yield: 65%, mp 203–
205 °C. 1H NMR (CDCl3) d: 2.82 (s, 3H), 3.93 (s, 3H), 3.96 (s, 6H),
5.83 (bs, 1H), 6.59 (d, J = 8.2 Hz, 1H), 7.11 (d, J = 8.2 Hz, 1H), 7.29
(m, 1H), 7.39 (s, 2H). Anal. Calcd for C19H18O6: C, 66.66; H, 5.30.
Found: C, 66.48; H, 5.11.
5.2.22. (5-Hydroxy-3-methylbenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanone (4w)
5.3.4. Synthesis of (6-methoxybenzofuran-2-yl)(3,4,5-
trimethoxyphenyl)methanol (4z)
The residue was chromatographed with EtOAc/petroleum ether
4:6 as eluent to give 4w as a yellow solid, yield: 53%, mp 181–
183 °C. 1H NMR (CDCl3) d: 2.59 (s, 3H), 3.93 (s, 3H), 3.96 (s, 6H),
7.01 (m, 1H), 7.06 (d, J = 6.4 Hz, 1H), 7.38 (d, J = 6.4 Hz, 1H), 7.41
(s, 1H), 7.42 (s, 2H). Anal. Calcd for C19H18O6: C, 66.66; H, 5.30.
Found: C, 66.50; H, 5.16.
To a cooled solution of 4d (1 mmol) in dioxane (5 mL) was
added NaBH4 (1 mmol), and then the mixture was allowed to stir
at room temperature under nitrogen for 2 h. The solvent was con-
centrated under reduced pressure, and aqueous hydrochloric acid
(1 M, 2 mL) was added to the residue. The mixture was extracted

6870
R. Romagnoli et al. / Bioorg. Med. Chem. 17 (2009) 6862–6871
with dichloromethane (2 ꢁ 10 mL) and the organic layer washed
with water (2 ꢁ 5 mL) and brine (5 mL), dried with Na2SO4 and
concentrated under reduced pressure to give a light yellow oil res-
idue. Purification by flash column chromatography (petroleum
ether/ethyl acetate 1:1) gave 4z as a white solid, which was recrys-
tallized from petroleum ether. Yield: 84%, mp 148–150 °C. 1H NMR
(CDCl3) d: 3.84 (s, 6H), 3.86 (s, 6H), 5.87 (bs, 1H), 6.47 (s, 1H), 6.57
(bs, 1H), 6.73 (s, 2H), 6.84 (dd, J = 8.6 and 2.4 Hz, 1H), 7.00 (d,
J = 2.4 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H). Anal. Calcd for C19H20O6:
C, 66.27; H, 5.85. Found: C, 66.04; H, 5.67.
leum ether 2:8 as eluent. Yield: 46%, brown solid, mp 124–
125 °C. 1H NMR (DMSO-d6) d: 3.88 (s, 6H), 3.93 (s, 3H), 4.04 (s,
3H), 6.92 (s, 1H), 7.00 (m, 2H), 7.73 (d, J = 9.2 Hz, 1H). Anal. Calcd
for C19H16O6: C, 67.05; H, 4.74. Found: C, 66.89; H, 4.59.
5.4. Cell growth inhibitory activity
Murine leukemia L1210, murine mammary carcinoma FM3A
and human T-lymphocyte Molt 4 and CEM and human cervix car-
cinoma (HeLa) cells were suspended at 300,000–500,000 cells/mL
of culture medium, and 100
lL of a cell suspension was added to
5.3.5. Preparation of 6-methoxy-2-(methoxy(3,4,5-
100 L of an appropriate dilution of the test compounds in wells
l
trimethoxyphenyl)methyl)benzofuran (4aa)
of 96-well microtiter plates. After incubation at 37 °C for two days,
cell number was determined using a Coulter counter. The IC50 value
was defined as the compound concentration required to inhibit cell
proliferation by 50%.
A mixture of 4z (86 mg, 0.26 mmol) and pyridinium p-toluen-
sulfonate (125 mg, 0.5 mmol) in a mixture of THF-MeOH (10 mL,
0.5:9.5) was stirred for 15 h in a sealed tube. The reaction mixture
was evaporated under vacuum, the crude mixture was dissolved
with dichloromethane (5 mL) and the organic phase was washed
with water (1 mL) and brine (1 mL), dried over Na2SO4 and concen-
trated under reduced pressure. The residue was chromatographed
with EtOAc/petroleum ether 3:7 as eluent to give 4aa as a white
solid, yield: 96%, mp 148–150 °C. 1H NMR (CDCl3) d: 3.46 (s, 3H),
3.84 (s, 3H), 3.85 (s, 3H), 3.87 (s, 6H), 5.29 (bs, 1H), 6.46 (d,
J = 1.0 Hz, 1H), 6.71 (s, 2H), 6.84 (dd, J = 8.4 and 2.2 Hz, 1H), 7.01
(d, J = 2.2 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H). Anal. Calcd for
C20H22O6: C, 67.03; H, 6.19. Found: C, 66.88; H, 6.07.
5.5. Effects on tubulin polymerization and on colchicine
binding to tubulin
Bovine brain tubulin was purified as described previously.19 To
evaluate the effect of the compounds on tubulin assembly
in vitro,14 varying concentrations were preincubated with 10
lM
tubulin in glutamate buffer at 30 °C and then cooled to 0 °C. After
addition of GTP, the mixtures were transferred to 0 °C cuvettes in a
recording spectrophotometer and warmed to 30 °C, and the assem-
bly of tubulin was observed turbidimetrically. The IC50 value was
defined as the compound concentration that inhibited the extent
of assembly by 50% after a 20 min incubation. The ability of the test
compounds to inhibit colchicine binding to tubulin was measured
5.3.6. Preparation of 2-(3,4,5-trimethoxybenzyl)-6-
methoxybenzofuran (4ab)
To a magnetically stirred suspension of 4z (0.5 mmol) in dichlo-
romethane (3 mL) at room temperature was added trifluoroacetic
as described,15 except that the reaction mixtures contained 1
tubulin, 5 M test compound.
M [3H]colchicine and 5
lM
acid (410
l
L, 5.5 mmol) and then triethylsilane (890
lL, 5.5 mmol).
l
l
After 15 min, the solvent was evaporated in vacuo, and the residue
was dissolved in dichloromethane (20 mL). The organic layer was
washed with an aqueous saturated solution of NaHCO3 (5 mL)
and brine (5 mL), dried (Na2SO4) and concentrated in vacuo. Flash
chromatography using ethyl acetate–petroleum ether 1:1, fur-
nished 4ab as a brown oil, yield: 94%. 1H NMR (CDCl3) d: 3.83 (s,
6H), 3.84 (s, 6H), 4.01 (s, 2H), 6.34 (d, J = 1.0 Hz, 1H), 6.51 (s, 2H),
6.82 (dd, J = 8.4 and 2.4 Hz, 1H), 6.98 (d, J = 2.4 Hz, 1H), 7.37 (d,
J = 8.5 Hz, 1H). Anal. Calcd for C19H20O5: C, 69.50; H, 6.14. Found:
C, 69.31; H, 5.95.
5.6. Flow cytometric analysis of cell cycle distribution
For details, see Ref. 9a.
5.7. Molecular modeling
All molecular modeling studies were performed on a MacPro
dual 2.66 GHz Xeon running Ubuntu 8. The tubulin structure was
downloaded from the PDB data bank (http://www.rcsb.org/ - PDB
code: 1SA0). Docking simulations were performed using PLANTS20
and results examined using ZODIAC.21 H-Bond and lipophilicity
maps were created using ZODIAC.
5.3.7. Synthesis of (2-bromo-3,4,5-trimethoxyphenyl)(6-
methoxybenzofuran-2-yl)methanone (7)
To a solution of 4d (342 mg, 1 mmol.) in acetonitrile (10 mL)
was added a mixture of benzoyl peroxide (48 mg, 0.2 mmol) and
NBS (214 mg, 1.2 mmol). The mixture was heated under reflux
for 2 h. The solvent was removed under reduced pressure, and
the residue dissolved in EtOAc (15 mL), which was washed succes-
sively with a saturated solution of NaHCO3 (5 mL), water (5 mL)
and brine (5 mL), dried (Na2SO4) and concentrated in vacuo. Puri-
fication by flash column chromatography (petroleum ether/ethyl
acetate 7:3) gave 7 as a white solid. Yield: 65%, mp 144–145 °C.
1H NMR (DMSO-d6) d: 3.83 (s, 6H), 3.85 (s, 3H), 3.86 (s, 3H), 6.99
(dd, J = 8.8 and 2.2 Hz, 1H), 7.15 (s, 1H), 7.36 (d, J = 2.4 Hz, 1H),
7.54 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H). ESI-MS m/z 421.0/423.0
[M+1]+/[M+3]+.
Acknowledgment
The authors would like to thank Dr. Alberto Casolari and Paolo
Orlandini for the technical assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.08.027.
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R. Romagnoli et al. / Bioorg. Med. Chem. 17 (2009) 6862–6871
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