Synthesis and biological evaluation of 2-(3′,4′,5′-trimethoxybenzoyl)-3-N,N-dimethylamino benzo[b]furan derivatives as inhibitors of tubulin polymerization

Article abstract of DOI:10.1016/j.bmc.2008.08.029

Molecules that target microtubules have an important role in the treatment of cancer. A new class of inhibitors of tubulin polymerization based on the 2-(3,4,5-trimethoxybenzoyl)-2-dimethylamino-benzo[b]furan molecular skeleton was synthesized and evaluat

Full text of DOI:10.1016/j.bmc.2008.08.029

Bioorganic & Medicinal Chemistry 16 (2008) 8419–8426
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 2-(30,40,50-trimethoxybenzoyl)-3-
N,N-dimethylamino benzo[b]furan derivatives as inhibitors of tubulin
polymerization
a,
b
a
a
Romeo Romagnoli a, , Pier Giovanni Baraldi , Taradas Sarkar , Maria Dora Carrion , Olga Cruz-Lopez ,
Carlota Lopez Cara a, Manlio Tolomeo c, Stefania Grimaudo d, Antonietta Di Cristina d,
Maria Rosaria Pipitone d, Jan Balzarini e, Roberto Gambari f, Lampronti Ilaria f, Roberto Saletti g,
Andrea Brancale g, Ernest Hamel e
*
*
a Dipartimento di Scienze Farmaceutiche, Università di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy
b 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
c Centro Interdipartimentale di Ricerca in Oncologia Clinica (C.I.R.O.C.) e Servizio AIDS, Università di Palermo, Palermo, Italy
d Dipartimento Biomedico di Medicina Interna e Specialistica, Università di Palermo, Palermo, Italy
e Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Minderbroedersstraat 10, B-3000 Leuven, Belgium
f Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, 44100 Ferrara, Italy
g The Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Molecules that target microtubules have an important role in the treatment of cancer. A new class of
inhibitors of tubulin polymerization based on the 2-(3,4,5-trimethoxybenzoyl)-2-dimethylamino-ben-
zo[b]furan molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition
of tubulin polymerization, and cell cycle effects. The most promising compound in this series was 2-
(3,4,5-trimethoxybenzoyl)-3-dimethylamino-6-methoxy-benzo[b]furan, which inhibits cancer cell
growth at nanomolar concentrations and interacts strongly with tubulin by binding to the colchicine site.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 27 June 2008
Revised 2 August 2008
Accepted 13 August 2008
Available online 17 August 2008
Keywords:
Microtubules
Tubulin polymerization
Colchicine binding site
Combretastatin A-4
Bioisosteric replacement
1. Introduction
of CA-4 analogues have been developed and evaluated in SAR
studies.4
Compounds that are able to interfere with the microtubule-
tubulin equilibrium in cells are useful in the treatment of human
diseases.1 The success of tubulin polymerization inhibitors as anti-
cancer agents has stimulated significant interest in the identifica-
tion of new compounds that may be more potent or more
selective in targeted tissues or tumors.
Among the microtubule depolymerizing agents, combretastatin
A-4 (CA-4, 1; Chart 1) is one of more studied compounds. CA-4, iso-
lated from the bark of the South African tree Combretum caffrum,2
strongly inhibits the polymerization of tubulin by binding to the
colchicine site.3 Because of its simple structure, a wide number
We have earlier reported three different series of 2-(30,40,50-
trimethoxybenzoyl)-3-amino benzoheterocyclic derivatives with
general structure 2–4, characterized by the presence of the
benzo[b]thiophene, 1H-indole, and 1-methylindole skeleton,
respectively.5,6 These compounds strongly inhibited tumor cell
growth and tubulin polymerization by binding to the colchicine
site of tubulin and caused G2/M phase arrest of the cell cycle.
The C-6 methoxy group plays an essential role for the inhibition
of tubulin polymerization within these series of molecules (com-
pounds 2a–4a).
As a part of our search for novel tubulin polymerization inhibi-
tors, we synthesized and evaluated the biological properties of a
new series of benzo[b]furan derivatives with general structure 5.
We based this synthesis on the bioisosteric replacement by furan
of either the thiophene or pyrrole moieties that characterized the
benzo[b]thiophene (2) and indole derivatives 3 and 4. Since the
* Corresponding authors. Tel.: +39 (0) 532 291290; fax: +39 (0) 532 291296
(R.R.); tel.: +39 (0) 532 291293; fax: +39 (0) 532 291296 (P.G.B.).
E-mail addresses: rmr@unife.it (R. Romagnoli), baraldi@unife.it (P.G. Baraldi).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.029

8420
R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 8419–8426
OCH3
H3CO
H3CO
OCH3
NH2
A
B
R
OCH3
CH3O
X
OH
OCH3
O
R=H, Me or OMe
X=S, benzo[b]thiophene derivatives 2
Combretastatin A-4 (CA-4), 1
X=NH, 1-H-indole derivatives 3
X=NMe, 1-methylindole derivatives 4
X=S, R=6-OMe, 2a
X=NH, R=6-OMe, 3a
X=NMe, R=6-OMe, 4a
OCH3
R5
N
H3CO
H3CO
OCH3
O
R1
R4
OCH3
R6
R2
R3
OCH3
O
O
5
R1
O
H3CO
R1-6=H; 5a
R1=OCH3, R2-6=H; 5b
OCH3
6
R2=OCH3, R1, 3-6=H, 5c
R3=OCH3, R1-2, 4-6=H; 5d
R4=OCH3, R1-3, 5, 6=H; 5e
R1=H, 6a
R1=OH, 6b
R1-4=H, R5, 6=CH3; 5f
R1=OCH3, R2-4=H, R5,6=CH3; 5g
R2=OCH3, R1, 3-4=H, R5,6=CH3; 5h
R3=OCH3, R1-2, 4-=H, R5,6=CH3; 5i
R4=OCH3, R1-3=H, R5, 6=CH3; 5j
R3=OCH3, R1-2, 4--5=H, R6=COCH3; 5k
R3=OCH3, R1-2, 4--5=H, R6=COCH2Cl; 5l
R3=OCH3, R1-2, 4--5=H, R6=COCH2Br; 5m
R3=OCH3, R1-2, 4--5=H, R6=COCH2I; 5n
Chart 1. Inhibitors of tubulin polymerization.
3,4,5-trimethoxybenzoyl substituent was demonstrated to be
essential for the bioactivity of derivatives 2–4, we maintained this
substituent at the 2-position of the benzo[b]furan skeleton
throughout the present investigation, and we examined the effects
of various substituents at the 3-position. This furnished three dif-
ferent small series of compounds.
The first series (derivatives 5a–e) was characterized by the
presence of a 3-amino group in the benzo[b]furan skeleton and
either no substituent (5a) or a methoxy group at each of the four
possible positions on the benzene ring (compounds 5b–e).
In the second series (compounds 5f–k), the 3-amino moiety
was replaced with a dimethylamino (5f) or acetamido group
(5k), plus a methoxy group at each of the four positions of the ben-
zene ring of the benzofuran moiety combined with the 3-dimeth-
ylamino group (5g–j) These compounds allowed us to determine
whether the 3-amino substituent could restrict the conformation
of the adjacent trimethoxybenzoyl moiety through an intramolec-
ular hydrogen bond with the carbonyl oxygen.
In the third series (5l–n), we examined haloacetyl amides at po-
sition 3 of the benzo[b]furan moiety. We were stimulated to do this
by previous studies in which the electrophilic nature of the halo-
acetyl moiety has been used in anticancer drug design.7,8 The alkyl-
ation of sulfhydryl groups of tubulin by iodoacetamide9 and the
disruption of microtubules by p-bromophenacyl bromide10 have
been reported.
We should note that previous studies have yielded a limited
series of tubulin inhibitors with the benzo[b]furan molecular
skeleton as the core structure. These compounds have general
structure 6, which incorporates the 3-(3,4,5-trimethoxybenzoyl)-
6-methoxybenzo[b]furan ring system.11 The 6-methoxy substitu-
ent is important for maximal activity and appears to correspond
to the 4-methoxy group in the B-ring of CA-4.4
2. Chemistry
Derivatives 5a–n were synthesized as shown in Scheme 1.
Refluxing 2-hydroxyaldehydes 7a–e and nitroethane in glacial ace-
tic acid in the presence of sodium acetate furnished the corre-
sponding nitriles 8a–e in good yields.12 The subsequent reaction
of 8a–e with 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone5 and
potassium carbonate in refluxing acetone yielded the cyclized 3-
amino benzo[b]furan derivatives 5a–e, which were transformed
into the corresponding 3-dimethylamino compounds 5f–j by treat-
ment with methyl iodide in DMF.
Acetylating or haloacetylating the 3-amino group of 5d with
acetyl chloride, chloroacetyl chloride, and bromoacetyl chloride
in the presence of pyridine provided the acetamide derivatives
5k–m, respectively. The iodoacetyl derivative 5n was prepared
from the bromoacetyl derivative 5m by an exchange reaction using
sodium iodide in N,N-dimethylacetamide.
3. Biological results and discussion
Table 1 summarizes the growth inhibitory effects of benzo[b]-
furan derivatives 5a–n against murine leukemia (L1210), murine
mammary carcinoma (FM3A), and human T-lymphoblastoid
(Molt/4 and CEM) cells, with CA-4 (1) and 2a–4a as reference
compounds.
Compound 5i was the most potent compound identified in this
study, inhibiting the growth of L1210, FM3A, Molt/4, and CEM can-

R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 8419–8426
8421
OCH3
OCH3
NH2
CHO
OH
CN
b
a
R1
R1
R1
OCH3
O
OH
O
R1=H, 5a
7a-e
8a-e
R1=4-OCH3, 5b
R1=5-OCH3, 5c
R1=6-OCH3, 5d
R1=7-OCH3, 5e
5a-e
R1=H, 7-8a
R1=6-OCH3, 7-8b
R1=5-OCH3, 7,8c
R1=4-OCH3, 7-8d
R1=3-OCH3, 7-8e
d, e and f
with compound 5d
c
X
OCH3
H3C
CH3
O
N
OCH3
O
OMe
R1
NH
OMe
OMe
OCH3
O
O
O
5g-j
R1=H, 5f
O
X=H, 5k
X=Cl, 5l
R1=4-OCH3, 5g
R1=5-OCH3, 5h
R1=6-OCH3, 5i
R1=7-OCH3, 5j
X=Br, 5m
g
X=I, 5n
Scheme 1. Reagents and conditions: (a) C2H5NO2, NaOAc; AcOH; (b) 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone, K2CO3, (CH3)2CO, reflux, 18 h, (c) Me1, NaH, DMF; (d)
CH3COCl, pyridine, CH2Cl2, rt; (e) ClCH2COCl, pyridine, CH2Cl2, rt; (f) BrCH2COBr, pyridine, CH2Cl2, rt; (g) Nal, CH3CON(CH3)2, rt.
four cell lines. From the point of view of SAR, the oxygen biostere
(5d) did not enhance activity relative to sulfur atom (2a), but did
Table 1
relative to the nitrogen atom (3a).
In vitro inhibitory effects of compounds 2–4a, 5a–n, and CA-4 (1) on the proliferation
With the exception of the C-4 methoxy derivative 5g, the meth-
of murine leukemia (L1210), murine mammary carcinoma (FM3A), and human T-
oxy substituent at C-5, C-6, or C-7 (5h–j, respectively) in the 3-
dimethylamino series increased antiproliferative activity relative
to both the unsubstituted 5f and their parent 3-amino counter-
parts 5c–e. This suggests that it would be worthwhile introducing
dimethylamino modification at the 3-amino group into the thio-
phene, indole, and methylindole series described previously.5,6
The antiproliferative activity of 5i was superior to that of 1H-in-
dole derivative 3a, comparable to that of 1-methylindole 4a, and
slightly less than that of benzo[b]thiophene 2a. Therefore, even
though the good activity of 5i required a further modification of
the 3-amino group, we conclude that there is validity to the idea
of bioisosteric equivalence between benzo[b]furan, 1-methylin-
dole, and benzo[b]thiophene in this class of compounds. Moreover,
comparing the structures of 5i with those of 6ab, we conclude that
the 3-dimethylamino group of 5i is analogous to the right-hand
substituted phenyl ring of 6ab (see Chart 1).
Changing the substituent at C-3 from the dimethylamino group
(5i) to an acetamido moiety (5k) caused only a minimal reduction
in antiproliferative activities. Consequently, 5k, like 5i, was supe-
rior in its antiproliferative properties to the amine, 5d.
Compound 5k was also more potent than 3-haloacetamido
derivatives 5l–n against all four cell lines. Of note in haloacetamido
series, increasing the size of the halogen atom resulted in increased
antiproliferative activity, with the order being I (5n) > Br (5m) > Cl
(5k). This order is also inversely related to the electron-withdraw-
ing properties of the halide atoms.
lymphocyte (Molt/4 and CEM) cells
a
Compound
L1210
IC50 (nM)
Molt4/C8
FM3A
CEM
5a
>10,000
>10,000
>10,000
1800 100
>10,000
>10,000
6700 100
>10,000
87 22
7700 400
>10,000
5b
5c
7500 900
>10,000
7900 2100
>10,000
5d
5e
430 40
>10,000
280 160
>10,000
140 20
4400 1400
2700 500
>10,000
5f
5g
>10,000
>10,000
8800 1300
>10,000
>10,000
1700 100
5h
5i
5j
5k
5l
5m
5n
2a
1400 110
1200 40
59
470 22
65
1100 100
94
5
5
48
4
78
1400 80
78
460 33
210 11
120 0.0
7.7 2.9
320 90
3
1200 900
100 0.00
1100 90
560 12
260 60
46 12
670 380
62 4.2
310 28
240 19
7
3
1100 90
480 15
170 30
39 16
86
10
5
7
3a
4a
CA-4 (1)
970 24
69 37
2.8 1.1
1600 30
97
42
630 10
57
16 1.4
3
6
7
71
1.9 1.6
5
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.
cer cell lines with IC50-values of 65, 59, 48, and 78 nM, respectively.
CA-4 (1) and, to a lesser extent, 2a were more potent as antiprolif-
erative agents than 5i with these four cell lines.
To confirm that the antiproliferative activities of these com-
pounds, like those of the benzothiophene and indole series,5,6 were
related to an interaction with the microtubule system, 5d, 5i, and
5k–n were evaluated for their inhibitory effects on tubulin
polymerization and on the binding of [3H]colchicine to tubulin
(Table 2).13,14 Comparing benzo-fused heterocyclic compounds
2a–4a and 5i, those containing either a sulfur (2a) or a nitrogen
(3a and 4a) in the benzoheterocyclic ring are less effective than
oxygen analogue 5i as an inhibitor of tubulin polymerization and
[3H] colchicine binding.
The results presented in Table 1 show that the location of the
methoxy group on the benzene portion of the benzo[b]furan moi-
ety plays a critical role in inhibition of cell growth, and the most
favorable position for the substituent was at C-6, as was observed
previously with the thiophene and indole series.5,6 In the series of
3-amino benzo[b]furan derivatives 5b–e, the 6-methoxy derivative
5d had the greatest antiproliferative activity, with IC50-values
ranging from 87 to 430 nM against the four cell lines. The 4-meth-
oxy analogue 5b was also over 10-fold less active than 5d in the

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R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 8419–8426
The order of inhibitory action on tubulin assembly was
trimethoxyphenyl group of the compounds reported here and the
corresponding ring in the DAMA-colchicine co-crystallized with
tubulin.19 The best correlation was observed with the MOE docking
program (r = 0.8), and the pose with the lowest RMSD value for
compound 5i is shown in Figure 2.
5i > 5d > 5k > 5n > 5l > 5m, which was consistent with the results
of the antiproliferative assays, except that 5d was more potent
than 5k. The most potent compound in this series was compound
5i, with an IC50 value of 0.9 lM. This is in agreement with 5i being
the compound with the greatest antiproliferative activity. Com-
pounds 5d and 5i were as active as CA-4 as inhibitors of tubulin
assembly, although both compounds were less active in their ef-
fects on cell growth.
From the docking results, it is possible to observe how the
methoxy group of the benzofuran ring of 5i can establish two
hydrogen bonds with Lys352 and Val181 of b-tubulin. This could
explain the difference in activity between 5i and the structurally
similar compounds 5f, 5g, 5h, and 5j, which were not able to estab-
lish the same interactions, given the different position of the meth-
oxy group on the benzofuran ring. Furthermore, compounds 5g, 5h,
and 5j were not docked successfully in the colchicine site by any of
the three software packages used.
In the colchicine binding studies, compounds 5d and 5i potently
inhibited the binding of [3H]colchicine to tubulin, since 73% and
76% inhibition, respectively, occurred with these agents at 1
lM
with colchicine at 5 M. These derivatives were slightly less potent
l
than CA-4, which in these experiments inhibited colchicine bind-
ing by 86%, but 5i was twofold more potent than its 1-methylin-
dole counterpart 4a.
4. Conclusions
Because molecules exhibiting effects on tubulin assembly
should cause the alteration of cell cycle parameters with preferen-
tial G2-M blockade, flow cytometry analysis was performed to
determine the effect of the most active compounds on K562 (hu-
man chronic myelogenous leukemia) cells. Cells were cultured
for 24 h in the presence of each compound at the IC50 value deter-
mined for 24 h of growth (5d = 120 nM, 5i = 68 nM, 5k = 85 nM,
5l = 400 nM, 5m = 212 nM, 5n = 115 nM). Analysis of sub-G0-G1
(apoptotic peak), G0-G1, S, and G2-M peaks revealed that the stud-
ied compounds caused somewhat different effects on cell cycle dis-
tribution (Fig. 1). While all six compounds caused an increase in
the proportion of cells in the G2-M peak, compounds 5d and 5l also
caused cells to accumulate in late S phase. In contrast, compound
5n caused the greatest percentage increase in the percentage of
cells in apoptotic cells (subG0-G1 peak), as well as substantial pro-
portion of cells in S phase.
We performed a series of molecular modeling studies on the
series of compounds reported here. In particular, we investigated
the use of three different docking software packages, Plants,15
the Surflex module implemented in Sybyl16 and MOE,17 in identify-
ing possible binding conformations for this family of compounds.
We also wanted to determine whether any combination of algo-
rithm/scoring function would generate a good correlation between
a calculated biological activity and the corresponding experimental
value for future use as a predictive model. The results obtained
with the different software packages were also rescored using
the scoring functions implemented in the CScore module of
Sybyl.16
In conclusion, the synthesis and biological evaluation of a new
class of synthetic antitubulin compounds based on the 2-(30,40,50-
trimethoxybenzoyl) benzo[b]furan skeleton is described. The re-
sults showed that compounds 5d and 5i, with a methoxy substitu-
tent at the C-6 position of the benzofuran ring, exhibited the best
antiproliferative activity in the 3-amino and 3-dimethylamino
benzo[b]furan series, respectively, while shifting the methoxy
group in both series to the C-3, C-5, or C-7 position resulted in ma-
jor losses in activity.
The dimethyl substitution on the amino group at the 3-position
of the benzo[b]furan core usually enhanced antiproliferative activ-
ity, with the dimethylamino derivatives 5f and 5h–j being more
potent than their amino counterparts 5a and 5c–e. In the series
of 3-haloacetamido derivatives 5l–n, the order of antiproliferative
activity was I > Br > Cl, but the greatest activity was observed with
the unsubstituted acetamido derivative 5k. Compound 5k was al-
most as active as 5i, the most potent antiproliferative agent in
the series.
The IC50-values for 5i in the cell lines examined ranged from 48
to 75 nM. The antiproliferative activity of 5i was equivalent to that
of the previously reported 1-methylindole derivative 4a and
slightly less than that of benzo[b]thiophene 2a. Compound 5i
strongly inhibited both the polymerization of tubulin and the bind-
ing of [3H]colchicine to tubulin, suggesting that 5i bound to tubulin
at a site overlapping the colchicine site. The antimitotic activity of
5i was demonstrated by flow cytometric analysis that showed that
5i had cellular effects typical of agents that bind to tubulin, causing
accumulation of cells in G2-M. We also were readily able to model
5i into the colchicine site, with excellent overlap with the colchici-
noid bound in X-ray crystal structure.
As noted previously, no correlation between the output of the
scoring functions and the experimental data was observed.18 How-
ever, a correlation was observed between the tubulin polymeriza-
tion assay results and the calculated RMSD value of the
5. Experimental
5.1. Chemistry
Table 2
Inhibition of tubulin polymerization and colchicine binding by compounds 2–4a, 5d,
5i, 5k–n, and CA-4
Compound
Tubulin assemblya
Colchicine bindingb
5.1.1. Materials and methods
IC50 SD (
l
M)
%
SD
2-Hydroxybenzaldehyde (7a), 2-hydroxy-6-methoxybenzalde-
hyde (7b), 2-hydroxy-5-methoxybenzaldehyde (7c), 2-hydroxy-4-
methoxybenzaldehyde (7d), 2-hydroxy-3-methoxybenzaldehyde
(7e), and 2-hydroxybenzonitrile (8a) are commercially available
and were used as received.
5d
5i
5k
5l
5m
5n
2a
3a
4a
1.1 0.1
0.90 0.0
1.6 0.0
2.0 0.1
2.3 0.5
1.8 0.1
1.3 0.1
>40
73
76
51
47
49
54
60
nd
38
86
5
5
7
5
2
1
0
1H NMR spectra were recorded on a Bruker AC 200 spectrome-
ter. Chemical shifts (d) are given in ppm upfield from tetramethyl-
silane as internal standard, and the spectra were recorded in
appropriate deuterated solvents, as indicated. Melting points
(mp) were determined on a Buchi–Tottoli apparatus and are uncor-
rected. All products reported showed 1H NMR spectra in agreement
with the assigned structures. Elemental analyses were conducted
4.6 0.1
1.2 0.1
5
3
CA-4 (1)
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 1 M, respectively. nd, not determined.
b
l

R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 8419–8426
8423
Fig. 1. Effects of compounds 5d (b), 5i (c), 5k (d), 5l (e), 5m (f), and 5n (g) on DNA content/cell following treatment of K562 cells for 24 h. The cells were cultured without
compound (Control, a) or with compound used at the concentration leading to 50% cell growth inhibition after 24 h of treatment. Cell cycle distribution was analyzed by the
standard propidium iodide procedure as described in Section 5. Sub-G0-G1 (A), G0-G1, S, and G2-M cells are indicated in the control panel.
Reaction courses and product mixtures were routinely monitored
by TLC on silica gel (precoated F254 Merck plates) and visualized
with aqueous KMnO4. Flash chromatography was performed using
230–400 mesh silica gel and the indicated solvent system. Organic
solutions were dried over anhydrous Na2SO4. Calcium chloride was
used in the distillation of DMF, and the distilled solvent was stored
over molecular sieves (3 Å).
5.1.2. General procedure A for the synthesis of 2-(30,40,50-
trimethoxybenzoyl)-3-amino benzo[b]furan (5a–e)
To a solution of 7a–e (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 solvent was evaporated, and the residue was dissolved
in a mixture of dichloromethane (15 mL) and water (5 mL). The or-
ganic layer was washed with brine, dried and evaporated to obtain
a residue, which was purified by flash column chromatography.
Then the final solid product was recrystallized from petroleum
ether.
5.1.2.1. (3-Aminobenzofuran-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (5a). Following general procedure A, the crude res-
idue purified by flash chromatography using ethyl acetate/petro-
leum ether 4:6 (v:v) as eluent furnished 5a as a yellow solid
(89% yield); mp 134–136 °C. 1H NMR (CDCl3) d: 3.95 (s, 3H), 3.97
(s, 6H), 6.04 (br s, 2H), 7.28 (t, J = 8.4 Hz, 1H), 7.43 (d, J = 8.4 Hz,
Fig. 2. Docking pose of 5i. DAMA-colchicine is represented in red.
by the Microanalytical Laboratory of the Chemistry Department of
the University of Ferrara. All reactions were carried out under an
inert atmosphere of dry nitrogen, unless otherwise described. Stan-
dard syringe techniques were applied for transferring dry solvents.

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R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 8419–8426
1H), 7.53 (t, J = 8.4 Hz, 1H), 7.60 (s, 2H), 7.64 (d, J = 8.4 Hz, 1H).
Anal. Calcd for C18H17NO5: C, 66.05; H, 5.23; N, 4.28. Found: C,
65.95; H, 5.11; N, 4.17.
5.1.3.2. (3-(Dimethylamino)-4-methoxybenzofuran-2-yl) (3,4,5-
trimethoxyphenyl)methanone (5g). Following general proce-
dure B, the crude residue purified by flash chromatography using
ethyl acetate/petroleum ether 3:7 (v:v) as eluent furnished 5g as
a yellow solid (43% yield); mp 148–150 °C. 1H NMR (CDCl3) d:
3.52 (s, 6H), 3.92 (s, 3H), 3.96 (s, 6H), 3.98 (s, 3H), 6.52 (d,
J = 7.8 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.8, 1H), 7.62 (s,
2H). Anal. Calcd for C21H23NO6: C, 65.44; H, 6.02; N, 3.63. Found:
C, 65.29; H, 5.88; N, 3.51.
5.1.2.2. (3-Amino-4-methoxybenzofuran-2-yl)(3,4,5-trimethoxy-
phenyl)methanone(5b). FollowinggeneralprocedureA,thecrude
residue purified by flash chromatography using ethyl acetate/petro-
leum ether 4:6 (v:v) as eluent furnished 5b as a yellow solid (78%
yield); mp 177–179 °C. 1H NMR (CDCl3) d: 3.91 (s, 3H), 3.94 (s, 3H),
3.97 (s, 6H), 6.52 (br s, 2H), 6.55 (d, J = 7.8 Hz, 1H), 6.98 (d, J = 7.8 Hz,
1H), 7.42 (t, J = 7.8, 1H), 7.57 (s, 2H). Anal. Calcd for C19H19NO6: C,
63.86; H, 5.35; N, 3.92. Found: C, 63.67; H, 5.21; N, 3.78.
5.1.3.3. (3-(Dimethylamino)-5-methoxybenzofuran-2-yl) (3,4,5-
trimethoxyphenyl)methanone (5h). Following general proce-
dure B, the crude residue purified by flash chromatography using
ethyl acetate/petroleum ether 3:7 (v:v) as eluent furnished 5h
as a yellow oil (44% yield). 1H NMR (CDCl3) d: 3.42 (s, 6H), 3.86
(s, 3H), 3.90 (s, 3H), 3.93 (s, 6H), 7.00 (s, 1H), 7.12 (d, J = 8.4 Hz,
1H), 7.34 (d, J = 8.4 Hz, 1H), 7.60 (s, 2H). Anal. Calcd for
5.1.2.3. (3-Amino-5-methoxybenzofuran-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (5c). Following general procedure A, the
crude residue purified by flash chromatography using ethyl ace-
tate/petroleum ether 4:6 (v:v) as eluent furnished 5c as a yellow so-
lid (76% yield); mp 125–127 °C. 1H NMR (CDCl3) d: 3.88 (s, 3H), 3.93
(s, 3H), 3.94 (s, 6H), 6.99 (s, 1H), 7.13 (d, J = 8.8 Hz, 1H), 7.26 (br s,
2H), 7.32 (d, J = 8.8 Hz, 1H), 7.58 (s, 2H). Anal. Calcd for
C21H23NO6: C, 65.44; H, 6.02; N, 3.63. Found: C, 65.33; H, 5.91;
N, 3.53.
C
3.80.
19H19NO6: C, 63.86; H, 5.35; N, 3.92. Found: C, 63.72; H, 5.14; N,
5.1.3.4. (3-(Dimethylamino)-6-methoxybenzofuran-2-yl) (3,4,5-
trimethoxyphenyl)methanone (5i). Following general proce-
dure B, the crude residue purified by flash chromatography using
ethyl acetate/petroleum ether 3:7 (v:v) as eluent furnished 5i as
a yellow oil (42% yield). 1H NMR (CDCl3) d: 3.38 (s, 6H), 3.88 (s,
3H), 3.94 (s, 3H), 3.96 (s, 6H), 6.92 (d, J = 8.0 Hz, 1H), 7.18 (d,
J = 8.0 Hz, 1H), 7.54 (s, 1H), 7.68 (s, 2H). Anal. Calcd for
5.1.2.4. (3-Amino-6-methoxybenzofuran-2-yl)(3,4,5-trimethoxy-
phenyl)methanone(5d). FollowinggeneralprocedureA, thecrude
residue purified by flash chromatography using ethyl acetate/petro-
leum ether 4:6 (v:v) as eluent furnished 5d as a yellow solid (77%
yield); mp 167–169 °C. 1H NMR (CDCl3) d: 3.89 (s, 3H), 3.94 (s, 3H),
3.97 (s, 6H), 6.87 (br s, 2H), 6.91 (d, J = 8.2 Hz, 1H), 7.48 (d, J = 8.2 Hz,
1H), 7.52 (s, 1H), 7.54 (s, 2H). Anal. Calcd for C19H19NO6: C, 63.86; H,
5.35; N, 3.92. Found: C, 63.77; H, 5.22; N, 3.74.
C21H23NO6: C, 65.44; H, 6.02; N, 3.63. Found: C, 65.31; H, 5.88;
N, 3.48.
5.1.3.5. (3-(Dimethylamino)-7-methoxybenzofuran-2-yl) (3,4,5-
trimethoxyphenyl)methanone (5j). Following general proce-
dure B, the crude residue purified by flash chromatography using
ethyl acetate/petroleum ether 3:7 (v:v) as eluent furnished 5j as a
yellow solid (93% yield); mp 148–150 °C. 1H NMR (CDCl3) d: 3.50
(s, 6H), 3.90 (s, 3H), 3.94 (s, 6H), 3.98 (s, 3H), 6.94 (d, J = 6.8 Hz,
1H), 7.18 (m, 2H), 7.62 (s, 2H). Anal. Calcd for C21H23NO6: C, 65.44;
H, 6.02; N, 3.63. Found: C, 65.34; H, 5.92; N, 3.52.
5.1.2.5. (3-Amino-7-methoxybenzofuran-2-yl)(3,4,5-trimethoxy-
phenyl)methanone(5e). Following generalprocedure A, the crude
residue purified by flash chromatography using ethyl acetate/petro-
leum ether 4:6 (v:v) as eluent furnished 2e as a yellow solid (87%
yield); mp 171–173 °C. 1H NMR (CDCl3) d: 3.93 (s, 3H), 3.97 (s, 6H),
3.99 (s, 3H), 6.84 (br s, 2H), 6.97 (d, J = 6.8 Hz, 1H), 7.21 (m, 2H), 7.66
(s, 2H). Anal. Calcd for C19H19NO6: C, 63.86; H, 5.35; N, 3.92. Found:
C, 63.59; H, 5.18; N, 3.80.
5.1.4. Synthesis of (3-acetylamino-6-methoxybenzofuran-2-yl)-
(3,4,5-trimethoxyphenyl)methanone (5k)
To a solution of 5d (1 mmol, 358 mg) and pyridine (3 mmol,
5.1.3. General procedure B for the synthesis of 2-(3,4,5-
trimethoxybenzoyl)-3-dimethylamino-benzo[b]furans (5f–j)
Sodium hydride (60% oil dispersion, 48 mg, 1 mmol) was care-
242
212
l
L) in dry dichloromethane (5 mL), acetyl chloride (3 mmol,
lL) was added at 0 °C. The reaction mixture was stirred for
2 h at room temperature, diluted with dichloromethane (5 mL),
washed with water (4 mL), dried over Na2SO4, and concentrated
in vacuo. The crude residue purified by flash chromatography using
ethyl acetate/petroleum ether 4:6 (v:v) as eluent furnished 5k as a
yellow solid (90% yield) after recrystallization from petroleum
ether; mp 172–173 °C. 1H NMR (CDCl3) d: 2.17 (s, 3H), 3.91 (s,
3H), 3.94 (s, 3H), 3.97 (s, 6H), 6.88 (s, 1H), 6.90 (d, J = 9.2 Hz, 1H),
7.53 (s, 2H), 8.47 (d, J = 9.2 Hz, 1H), 10.9 (s, 1H). Anal.
(C21H21NO7): C, H, N. Anal. Calcd for C21H21NO7: C, 63.15; H,
5.30; N, 3.51. Found: C, 63.02; H, 5.18; N, 3.38.
fully added to an ice-cooled solution of CH3I (93 lL, 1.5 mmol)
and 5a–e (0.56 mmol) in 2 mL of anhydrous DMF. The reaction
vessel was sealed and the mixture was stirred at 40 °C for 48 h.
After this period, CH3I (124 lL, 2 mmol) was added, and after
72 h the reaction mixture was diluted with cold water (1 mL)
and extracted with CH2Cl2 (3 ꢀ 5 mL). The combined organic ex-
tracts were washed with water (2 mL) and brine, dried and con-
centrated in vacuo. The resulting residue was purified by flash
chromatography using a mixture of ethyl acetate/petroleum ether
as eluent. The final solid product was recrystallized from petro-
leum ether.
5.1.5. Synthesis o(f3-chloroacetylamino-6-methoxybenzofuran-2-
yl)(3,4,5-trimethoxyphenyl)methanone (5l)
5.1.3.1. (3-(Dimethylamino)benzofuran-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (5f). Following general procedure B, the
crude residue purified by flash chromatography using ethyl ace-
tate/petroleum ether 3:7 (v:v) as eluent furnished 5f as a yellow oil
(46% yield). 1H NMR (CDCl3) d: 3.32 (s, 6H), 3.93 (s, 3H), 3.96 (s, 6H),
7.30 (t, J = 8.2 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.56 (t, J = 8.2 Hz, 1H),
7.64 (s, 2H), 7.68 (d, J = 8.2 Hz, 1H). Anal. (C20H21NO5): C, H, N. Anal.
(C19H19NO6): C, H, N. Anal. Calcd for C20H21NO5: C, 67.45; H, 5.96;
N, 3.94. Found: C, 67.45; H, 5.82; N, 3.83.
To a solution of 5d (1 mmol, 358 mg) and pyridine (3 mmol,
242
lL) in dry dichloromethane (5 mL), chloroacetyl chloride
(3 mmol, 239
lL) was added at 0 °C. The reaction mixture was stir-
red for 1 h at room temperature, diluted with dichloromethane
(10 mL), washed with water (5 mL), dried over Na2SO4, and con-
centrated in vacuo. The crude residue purified by flash chromatog-
raphy using ethyl acetate/petroleum ether 3:7 (v:v) as eluent
furnished 5l as a green solid (>95% yield) after recrystallization
from petroleum ether; mp 123–124 °C. 1H NMR (CDCl3) d: 3.91

R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 8419–8426
8425
(s, 3H), 3.97 (s, 9H), 4.31 (s, 2H), 6.91 (s, 1H), 6.95 (d, J = 9.2 Hz, 1H),
7.55 (s, 2H), 8.47 (d, J = 9.2 Hz, 1H), 11.7 (s, 1H). Anal. Calcd for
21H20ClNO7: C, 58.14; H, 4.65; N, 3.23. Found: C, 58.01; H, 4.48;
5.4. Flow cytometric analysis of cell cycle distribution
C
The effects of the most active compounds of the series on cell
cycle distribution were studied on K562 cells (myeloblastic leuke-
mia) by flow cytometric analysis after staining with propidium io-
dide. Cells were exposed for 24 h to each compound used at a
concentration corresponding to the IC50 determined after a 24 h
incubation. After treatment, the cells were washed once in ice-cold
PBS and resuspended at 1 ꢀ 106 per mL in a hypotonic fluoro-
N, 3.11.
5.1.6. Synthesis of (3-bromoacetylamino-6-methoxy-
benzofuran-2-yl)(3,4,5-trimethoxyphenyl)methanone (5m)
To a solution of 5d (1 mmol, 358 mg) and pyridine (3 mmol,
242
(3 mmol, 250
l
L) in dry dichloromethane (5 mL), bromoacetyl chloride
L) was added at 0 °C. After 3 h at the same tempera-
l
chrome solution containing propidium iodide (Sigma) at 50 lg/
ture, the reaction mixturewas diluted with dichloromethane (5 mL),
washed with water (5 mL), dried over Na2SO4, and concentrated in
vacuo. The crude residue purified by flash chromatography using
ethyl acetate/petroleum ether 3:7 (v:v) as eluent furnished 5 m as
a green solid (78% yield) after recrystallization from petroleum
ether; mp 99–100 °C. 1H NMR (CDCl3) d: 3.89 (s, 3H), 3.92 (s, 6H),
3.97 (s, 3H), 4.13 (s, 2H), 6.91 (s, 1H), 6.94 (d, J = 9.2 Hz, 1H), 7.54
(s, 2H), 8.45 (d, J = 9.2 Hz, 1H), 11.6 (s, 1H). Anal. Calcd for
mL in 0.1% sodium citrate plus 0.03% (v/v) nonidet P-40 (Sigma).
After a 30 min incubation, the fluorescence of each sample was
analyzed as single-parameter frequency histogram, using a FAC-
Scan flow cytometer (Becton–Dickinson, San Jose, CA). The distri-
bution of cells in the cell cycle was analyzed with the ModFit LT3
program (Verity Software House, Inc.).
5.5. Molecular modeling
C21H20BrNO7: C, 52.73; H, 4.21; N, 2.93. Found: C, 52.62; H, 4.04;
N, 2.78.
All molecular modelingstudies were performed on a MacPro dual
2.66 GHz Xeon running Ubuntu 7. The tubulin structure was down-
loaded from the PDB (http://www.rcsb.org/—PDB code: 1SA0).19
Hydrogen atoms were added to the protein using Molecular Operat-
ing Environment (MOE) 2006.0817 and minimized using the
MMFF94x forcefield until a RMSD gradient of 0.05 kcal molꢁ1 Åꢁ1
was reached. The docking simulations with MOE were performed
using the Alpha Triangle placement method and the London dG scor-
ing method. Plants15 was used from the graphical interface included
in ZODIAC21 with the default settings. The Surflex module included
in Sybyl 7.316 was used with the default settings with a ligand-based
(DAMA-colchicine) protomodel. The results obtained witheach soft-
ware package were then rescored using the CScore module in Sybyl
7.3. TheRMSD of thetrimethoxyphenyl moietyfor eachof theresults
obtained was calculated in comparison with ring A of the colchicine
analogue.22
5.1.7. Synthesis of (3-iodoacetylamino-6-methoxybenzofuran-
2-yl)(3,4,5-trimethoxyphenyl)methanone (5n)
A mixture of 5m (1 mmol, 478 mg) and NaI (10 mmol, 1.5 g) in
N,N-dimethylacetamide (5 mL) was stirred at room temperature
for 18 h. N,N-dimethylacetamide was evaporated under reduced
pressure, followed by addition of dichloromethane (15 mL) and a
solution of Na2S2O3 (10%, 5 mL). The organic layer was washed
with water (5 mL), brine (5 mL), and dried over Na2SO4. After re-
moval of the solvent under reduced pressure, the crude residue
purified by flash chromatography using ethyl acetate/petroleum
ether 4:6 (v:v) as eluent furnished 5n as a yellow solid (58% yield)
after recrystallization from petroleum ether; mp 140–141 °C. 1H
NMR (CDCl3) d: 3.72 (s, 2H), 3.90 (s, 3H), 3.97 (s, 6H), 3. 98 (s,
3H), 6.89 (s, 1H), 6.92 (d, J = 8.8 Hz, 1H), 7.54 (s, 2H), 8.45 (d,
J = 8.8 Hz, 1H), 11.5 (s, 1H). Anal. Calcd for C21H20INO7: C, 48.02;
H, 3.84; N, 2.67. Found: C, 47.88; H, 3.74; N, 2.56.
References and notes
1. (a) Kiselyov, A.; Bulakin, K. V. Anti-Cancer Agents Med. Chem. 2007, 7, 189; (b)
Nagle, A.; Hur, W.; Gray, N. S. Curr. Drug Targets 2006, 7, 305; (c) Pasquier, E.;
Andrè, N.; Braguer, D. Curr. Cancer Drug Targets 2007, 7, 566.
5.2. Cell growth inhibitory activity
2. (a) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall,
D. Experentia 1989, 45, 209; (b) Chaplin, D. J.; Horsman, M. R.; Siemann, D. W.
Curr. Opin. Invest. Drugs 2006, 7, 522.
3. Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Biochemistry 1989, 28, 6984.
4. Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. J. Med.
Chem. 2006, 49, 3033.
5. Romagnoli, R.; Baraldi, P. G.; Carrion, M. D.; Lopez Cara, C.; Preti, D.; Fruttarolo,
F.; Pavani, M. G.; Tabrizi, M. A.; Tolomeo, M.; Grimaudo, S.; Di Antonella, C.;
Balzarini, J.; Hadfield, J. A.; Brancale, A.; Hamel, E. J. Med. Chem. 2007, 50, 2273.
6. Romagnoli, R.; Baraldi, P. G.; Sarkar, T.; Carrion, M. D.; Lopez-Cara, C.; Cruz-Lopez,
O.; Preti, D.; Tabrizi, M. A.; Tolomeo, M.; Grimaudo, S.; Di Cristina, A.; Zonta, N.;
Balzarini, J.; Brancale, A.; Hsieh, H. P.; Hamel, E. J. Med. Chem. 2008, 51, 1464.
7. Baker, B.; Devan, B. P. J. Am. Chem. Soc. 1989, 111, 2700.
Murine leukemia L1210, murine mammary carcinoma FM3A,
and human T-lymphocyte Molt 4 and CEM cells were suspended
at 300,000–500,000 cells/mL of culture medium, and 100
lL of a
cell suspension was added to 100 L of an appropriate dilution of
l
the test compounds in wells of 96-well microtiter plates. After
incubation at 37 °C for two (L1210 and FM3A) or three (Molt 4
and CEM) days, cell number was determined using a Coulter coun-
ter. The IC50 value was defined as the compound concentration re-
quired to inhibit cell proliferation by 50%.
8. (a) Jiang, J.-D.; Roboz, J.; Weisz, I.; Deng, L.; Ma, L.; Holland, J. F.; Bekesi, J. G.
Anti-Cancer Drug Des. 1998, 13, 735; (b) Song, D.-Q.; Wang, Y.; Wu, L.-Z.; Yang,
P.; Wang, Y.-M.; Gao, L.-M.; Li, Y.; Qu, J.-R.; Wang, Y.-H.; Li, Y.-H.; Du, N. N.;
Han, Y.-X.; Zhang, Z.-P.; Jiang, J. D. J. Med. Chem. 2008, 51, 3094.
9. Luduena, R. F.; Roach, M. C. Pharmacol. Ther. 1991, 49, 133.
5.3. Effects on tubulin polymerization and on colchicine
binding to tubulin
Bovine brain tubulin was purified as described previously.20 To
evaluate the effect of the compounds on tubulin assembly
10. Hargreaves, A. J.; Glazier, A. P.; Flaskos, J.; Mullins, F. H.; McLean, W. G.
Biochem. Pharmacol. 1994, 47, 1137.
in vitro,13 varying concentrations were preincubated with 10
lM
11. Flynn, B. L.; Hamel, E.; Jung, M. K. J. Med. Chem. 2002, 45, 2670.
12. For the synthesis of 8b–e, see the procedure reported in the article: Karmarkar,
S. N.; Kelkar, S. L.; Wadia, M. S. Synthesis 1985, 510; For the characterization of
the 2-hydroxy-6-methoxybenzonitrile (8b) see: Hwu, J. R.; Wong, F. F.; Huang,
J.-J.; Tsay, S.-C. J. Org. Chem. 1997, 62, 4097; For the characterization of the 2-
hydroxy-5-methoxybenzonitrile (8c) and 2-hydroxy-4-methoxybenzonitrile
(8d) see: Adachi, M.; Sugasawa, T. Synth. Commun. 1990, 20, 71; For the
characterization of the 2-hydroxy-3-methoxybenzonitrile (8e) see: Dewan, S.
K.; Singh, R.; Kumar, A. Synth. Commun. 2004, 34, 2025.
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
assembly of tubulin was observed turbidimetrically. The IC50 va-
lue was defined as the compound concentration that inhibited
the extent of assembly by 50% after a 20 min incubation. The abil-
ity of the test compounds to inhibit colchicine binding to tubulin
was measured as described,14 except that the reaction mixtures
13. Hamel, E. Cell Biochem. Biophys. 2003, 38, 1.
14. Verdier-Pinard, P.; Lai, J.-Y.; Yoo, H.-D.; Yu, J.; Marquez, B.; Nagle, D. G.; Nambu,
M.; White, J. D.; Falck, J. R.; Gerwick, W. H.; Day, B. W.; Hamel, E. Mol.
Pharmacol. 1998, 53, 62.
contained
1 lM tubulin, 5 lM 1 lM test
[3H]colchicine, and
compound.
15. Korb, O.; Stützle, T.; Exner, T. E. Swarm Intell. 2007, 1, 115.

8426
R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 8419–8426
16. Tripos SYBYL 7.3; Tripos Inc., 1699 South Hanley Road, St. Louis, MO 63144,
USA. http://www.tripos.com.
19. Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.; Sobel, A.;
Knossow, M. Nature 2004, 428, 198.
17. Molecular Operating Environment (MOE 2006.08). Chemical Computing
Group, Inc., Montreal, Que., Canada. http://www.chemcomp.com.
18. De Martino, G.; Edler, M. C.; La Regina, G.; Coluccia, A.; Barbera, M. C.; Barrow,
D.; Nicholson, R. I.; Chiosis, G.; Brancale, A.; Hamel, E.; Artico, M.; Silvestri, R.
J. Med. Chem. 2006, 49, 947.
20. Hamel, E.; Lin, C. M. Biochemistry 1984, 23, 4173.
21. Zodiac 0.3.5b. http://www.zeden.org.
22. Code ‘fragment_rmsd.svl’ obtained from SLV Exchange website http://
svl.chemcomp.com.
Canada.
Chemical
Computing
Group,
Inc.,
Montreal,