Synthesis and Antitumor Molecular Mechanism of Agents Based on Amino 2-(3',4',5'-Trimethoxybenzoyl)benzo[b]furan: Inhibition of Tubulin and Induction of Apoptosis

Article abstract of DOI:10.1002/cmdc.201100279

Induction of apoptosis is a promising strategy that could lead to the discovery of new molecules active in cancer chemotherapy. This property is generally observed when cells are treated with agents that target microtubules, dynamic structures that play a

Full text of DOI:10.1002/cmdc.201100279

DOI: 10.1002/cmdc.201100279
Synthesis and Antitumor Molecular Mechanism of Agents
Based on Amino 2-(3’,4’,5’-Trimethoxybenzoyl)-
benzo[b]furan: Inhibition of Tubulin and Induction of
Apoptosis
Romeo Romagnoli,*[a] Pier Giovanni Baraldi,[a] Carlota Lopez-Cara,[a] Olga Cruz-Lopez,[a]
Maria Dora Carrion,[a] Maria Kimatrai Salvador,[a] Jaime Bermejo,[b] Sara Estꢀvez,[c]
Francisco Estꢀvez,[c] Jan Balzarini,[d] Andrea Brancale,[e] Antonio Ricci,[e] Longchuan Chen,[f]
Jae Gwan Kim,[g] and Ernest Hamel[h]
Induction of apoptosis is a promising strategy that could lead
to the discovery of new molecules active in cancer chemother-
apy. This property is generally observed when cells are treated
with agents that target microtubules, dynamic structures that
play a crucial role in cell division. Small molecules such as ben-
zo[b]furans are attractive as inhibitors of tubulin polymeri-
zation. A new class of inhibitors of tubulin polymerization
based on the 2-(3’,4’,5’-trimethoxybenzoyl)benzo[b]furan mo-
lecular skeleton, with the amino group placed at different posi-
tions on the benzene ring, were synthesized and evaluated for
antiproliferative activity, inhibition of tubulin polymerization,
and cell-cycle effects. The methoxy substitution pattern on the
benzene portion of the benzo[b]furan moiety played an impor-
tant role in affecting antiproliferative activity. In the series of 5-
amino derivatives, the greatest inhibition of cell growth oc-
curred if the methoxy substituent is placed at the C6 position,
whereas C7 substitution decreases potency. The most promis-
ing compound in this series is 2-(3’,4’,5’-trimethoxybenzoyl)-3-
methyl-5-amino-6-methoxybenzo[b]furan (3h), which inhibits
cancer cell growth at nanomolar concentrations (IC50 =16–
24 nm), and interacts strongly with tubulin by binding to the
colchicine site. Sub-G1 apoptotic cells in cultures of HL-60 and
U937 cells were observed by flow cytometric analysis after
treatment with 3h in a concentration-dependent manner. We
also show that compound 3h induces apoptosis by activation
of caspase-3, -8, and -9, and this is associated with cytochro-
me c release from mitochondria. The introduction of an a-bro-
moacryloyl group increased antiproliferative activity with re-
spect to the parent amino derivatives.
Introduction
Apoptosis, programmed cell death, plays a critical role in
normal cell development and is a well-regulated mechanism to
eliminate damaged and dysfunctional cells.[1] This process was
recently recognized as an important phenomenon for success-
ful cancer chemotherapy[2] and identification of compounds
that activate apoptosis is recognized as a promising strategy
for the discovery of potential anticancer agents.[3]
Antimitotic agents are a major class of cytotoxic drugs for
cancer treatment, and tubulin is the target for numerous small
natural and synthetic molecules that inhibit the formation of
[a] Dr. R. Romagnoli, Dr. P. G. Baraldi, Dr. C. Lopez-Cara, Dr. O. Cruz-Lopez,
Prof. M. D. Carrion, Dr. M. Kimatrai Salvador
Dipartimento di Scienze Farmaceutiche
[e] Dr. A. Brancale, Dr. A. Ricci
The Welsh School of Pharmacy
Cardiff University, Cardiff, CF10 3XF (UK)
Universitꢀ di Ferrara, 44100 Ferrara (Italy)
E-mail: rmr@unife.it
[f] Prof. L. Chen
Department of Pathology
[b] Prof. J. Bermejo
VA Medical Center, Long Beach, CA 90822 (USA)
Instituto de Productos Naturales y Agrobiologꢁa – C.S.I.C.
Instituto Universitario de Bio-Orgꢂnica “Antonio Gonzꢂlez”
Universidad de La Laguna, Instituto Canario de Investigaciꢃn del Cꢂncer,
38206 La Laguna, Tenerife (Spain)
[g] Dr. J. G. Kim
Department of Information and Communications
Gwangju Institute of Science and Technology, Gwangju, 500-712 (Korea)
[h] Dr. E. Hamel
[c] Dr. S. Estꢄvez, Prof. F. Estꢄvez
Screening Technologies Branch, Developmental Therapeutics Program
Division of Cancer Treatment and Diagnosis
National Cancer Institute at Frederick
Departamento de Bioquꢁmica
Universidad de Las Palmas de Gran Canaria
Unidad Asociada al C.S.I.C. and Instituto Canario de Investigaciꢃn del
Cꢂncer, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria (Spain)
National Institutes of Health, Frederick, MD 21702 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100279.
[d] Prof. J. Balzarini
Rega Institute for Medical Research
Laboratory of Virology and Chemotherapy, 3000 Leuven (Belgium)
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the mitotic spindle.[4] The microtubule system of eukaryotic
cells is a critical element in a variety of essential cellular pro-
cesses in addition to mitotic spindle assembly, including deter-
mination and maintenance of cell shape, regulation of motility,
cell signaling, secretion, and intracellular transport.[5]
As a part of our continuing search for synthetic small mole-
cule tubulin inhibitors, we previously reported a series of me-
thoxy-substituted
2-(3’,4’,5’-trimethoxybenzoyl)-3-aminoben-
zo[b]furan derivatives, with general structure 2, as a new class
of antimitotic agents.[9a] In this series of compounds, potent ac-
tivity was highly dependent on the presence and position of
four methoxy substituents.[13] A fairly dramatic difference was
observed between C4/5 and C6/7-methoxy substituted com-
pounds. The greatest activity occurred when the methoxy
group was located at the 6- or 7-position, whereas 4- and 5-
methoxy derivatives were inactive. Compound 2a, with a me-
thoxy group at the 6-position, displayed potent growth inhibi-
tion of different cancer cell lines. This agent inhibited tubulin
polymerization and caused G2/M phase arrest of the cell cycle.
These results prompted us to study this class of compounds in
further detail.
Among the natural microtubule depolymerizing agents,
combretastatin A-4 (CA-4, 1a, Figure 1) is one of the more
studied compounds. CA-4, isolated from the bark of the South
African tree Combretum caffrum,[6] strongly inhibits the poly-
merization of tubulin by binding to the colchicine site.[7] CA-4
inhibits cell growth at low- to mid-nanomolar concentrations.
The water-soluble sodium phosphate prodrug of CA-4 (CA-4P,
fosbretabulin, or Zybrestat, 1b) has yielded promising results
in human clinical trials.[8] Because of its structural simplicity,
along with its ability to selectivity damage tumor neovascula-
ture, numerous CA-4 analogues have been developed and
evaluated in structure–activity relationship (SAR) studies.[9]
The 3’-hydroxy group of the CA-4 B ring can be replaced by
an amine to furnish the 3’-amino derivative 1c, named AVE-
8063 or AC-7739, with no substantial loss of activity. This com-
pound is a potent inhibitor of tubulin assembly and also
showed significant cytotoxicity against human cancer cell
lines.[10] The corresponding amido serine prodrugs (1d, AVE-
8062 or Ambrabulin) and CA-4P are potent vascular disrupting
agents (VDA) and have been demonstrated to cause vascular
shutdown in established tumors in vivo.[11] AVE-6062 was found
to have more potent activity compared with CA-4P and is cur-
rently undergoing phase III clinical trials for the treatment of
solid tumors.[12]
Herein we describe the synthesis and SAR of new 2-(3’,4’,5’-
trimethoxybenzoyl)benzo[b]furan derivatives, some of which
are potent antitubulin agents. In this series, the 3’,4’,5’-trime-
thoxyphenyl of the 2-benzoyl moiety was kept unchanged be-
cause it is the characteristic structural requirement for activity
in numerous inhibitors of tubulin polymerization, such as col-
chicine, CA-4, and podophyllotoxin.[14]
In the first series of new derivatives described herein, the
amino group of the compound with general structure 2 was
moved from the 3-position to each of the four possible posi-
tions of the benzene portion of the benzo[b]furan skeleton, to
obtain compounds 3a–e, with the concomitant presence of an
additional methoxy group at the 5- (3 f), 6- (3g and 3h), or 7-
(3i and 3j) positions.
With the aim of optimizing the potency of this first class of
compounds, we also synthesized a second series of molecules
(4a–i), characterized by the presence of a Michael acceptor,
corresponding to an a-bromoacryloyl alkylating moiety of low
chemical reactivity, an unusual feature for cytotoxic com-
pounds.[15] The reactivity of the a-bromoacryoyl moiety has
been hypothesized to be based on a first-step Michael-type
nucleophilic attack, followed by a further reaction of the
former vinylic bromo substituent alpha to the carbonyl, lead-
ing successively either to a second nucleophilic substitution or
to beta elimination.[16]
An approach to increase water solubility is the preparation
of amino acid prodrugs, that release the parent drug following
enzymatic cleavage.[17] In the series of compounds described
herein, the presence of an amino moiety provides a site for
preparation of amino acid prodrugs, similar to what was suc-
cessfully done in the case of AVE-8062. Thus, we synthesized
the glycine amino acid prodrug of compound 3h, compound
5.
Results and Discussion
Chemistry
The nitro 2-(3’,4’,5’-trimethoxybenzoyl)benzo[b]furan deriva-
tives 7a–j were synthesized in good yield by a “one-step” cycli-
zation reaction of the corresponding, variously substituted ni-
Figure 1. Lead structures of tubulin polymerization inhibitors.
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Benzo[b]furan-Based Inhibitors
human T-lymphoblastoid (Molt/4 and CEM), and human cervix
carcinoma (HeLa) cells, with CA-4 (1) and 2a employed as ref-
erence compounds. The 3-methyl-5-amino-6-methoxy deriva-
tive 3h was the most active compound identified in this study,
inhibiting the growth of L1210, FM3A, Molt/4, CEM, and HeLa
cells with IC50 values of 19, 24, 22, 22, and 16 nm, respectively.
Compound 3h was twofold more potent than CA-4 against
FM3A cells. The two compounds were equipotent against
Molt/4 cells, but 3h was much less active than CA-4 against
L1210, CEM, and HeLa cells. Moving the amino group from the
3- to the 5-position (compound 2a versus 3g) led to increased
activity against L1210, FM3A, and Molt/4 cells, whereas these
derivatives had similar activity against CEM cells. These effects
were more evident in the 3-methyl counterpart 3h, which was
4–20-fold more active than 2a. Thus, the 3-amino position
cannot be considered essential for activity. SAR information de-
rived from comparing the 3-unsubstituted derivatives 3b, g,
and i with their 3-methyl congeners 3c, h, and j showed that
introduction of the methyl almost always increased antiproli-
ferative activity against these five cell lines.
Scheme 1. Synthesis of compounds 3a–j, 4a–i, and 5. Reagents and condi-
tions: a) 1-(3,4,5-trimethoxyphenyl)-2-bromoethanone, K2CO3, (CH3)2CO,
reflux, 18 h; b) Fe, HCl (37% in H2O), EtOH, reflux, 3 h; c) a-bromoacrylic acid,
EDCI, HOBt, DMF, RT, 18 h; d) N-Boc-glycine, EDCI, HOBt, DMF, RT, 12 h;
e) 3m HCl in EtOAc, RT, 3 h.
In the series of amino monosubstituted derivatives 3a, 3b,
3d, and 3e, only the 4-amino isomer 3a exhibited antiprolifer-
ative activity below 10 mm (IC50 =2.2–9.2 mm). Starting from the
4-amino derivative 3b, addition of a C3 methyl group to fur-
nish 3c, caused an increase in activity. The activity of these
compounds was further influenced by the presence and posi-
tion of a methoxy group in the benzene portion of the ben-
zo[b]furan skeleton. In comparing the C6 and C7 methoxy de-
rivatives, the greatest activity occurred with the methoxy
trosalicylaldehydes 6a, b, d, e, g, and i or 2-hydroxyacetophe-
nones 6c, f, h, and j with 2-bromo-1-(3’,4’,5’-trimethoxypheny-
l)ethanone and anhydrous potassium carbonate in acetone at
reflux (Scheme 1).[18] Subsequent reduction of the aromatic
nitro group with iron in a mixture of 37% HCl in water and
ethanol at reflux afforded the
corresponding amino derivatives
3a–j. The hybrid compounds
Table 1. In vitro inhibitory effects of compounds 2a, 3a–j, 4a–i, and CA-4 against the proliferation of murine
leukemia (L1210), murine mammary carcinoma (FM3A), human T-lymphocyte (Molt/4 and CEM), and human
cervix carcinoma (HeLa) cells.
4a–i were prepared in accepta-
ble yields by the condensation
of a-bromoacrylic acid with
amino benzo[b]furan derivatives
4a–f and 4h, i, and j using an
excess (2 equiv) of both EDCI
and 1-HOBt in dry DMF as sol-
vent at room temperature and
with identical reaction times
(18 h). The glycine prodrug 5
was synthesized by coupling the
amino derivative 3h with N-Boc-
glycine using EDCI and HOBt as
coupling agents, followed by
cleavage of the N-Boc protecting
group with 3m HCl in EtOAc.
Compd
IC50 [nm][a]
L1210
FM3A/0
Molt4/C8
CEM/0
HeLa
3a
3b
3c
3d
3e
3 f
3g
3h
3i
6700Æ600
9200Æ800
ND
5400Æ1200
2200Æ600
>10000
>10000
>10000
>10000
>10000
2500Æ100
>10000
>10000
>10000
3600Æ150
>10000
>10000
>10000
ND
2400Æ400
>10000
>10000
>10000
5500Æ530
7500Æ200
>10000
>10000
>10000
>10000
>10000
42Æ3.8
73Æ24
73Æ2
66Æ6.7
22Æ4
870Æ20
360Æ20
ND
230Æ1
ND
ND
1100Æ90
ND
59Æ4.1
19Æ2
600Æ90
67Æ4
24Æ6
560Æ90
140Æ13
200Æ90
320Æ30
520Æ29
1200Æ90
780Æ62
5900Æ380
960Æ78
250Æ15
1100Æ100
10Æ8
22Æ5
450Æ10
120Æ10
110Æ20
390Æ30
310Æ80
600Æ37
970Æ55
3200Æ240
340Æ20
240Æ38
770Æ15
20Æ2
16Æ1
370Æ30
490Æ54
1000Æ80
1100Æ90
430Æ90
1200Æ80
1000Æ100
5900Æ900
1000Æ100
780Æ54
1400Æ120
14Æ1
3j
4a
4b
4c
4d
4e
4 f
4g
4h
4i
78Æ2.8
150Æ8
320Æ17
420Æ90
800Æ51
1200Æ40
430Æ23
120Æ14
390Æ20
14Æ1
Biology
ND
300Æ40
920Æ91
18Æ1
140Æ20
16Æ1.4
Table 1 summarizes the growth
inhibitory effects of 2-(3’,4’,5’-tri-
methoxy)benzo[b]furan deriva-
tives 3a–j, 4a–i, and 5 against
murine leukemia (L1210), murine
mammary carcinoma (FM3A),
5
2a
CA-4
430Æ40
2.8Æ1.1
280Æ16
42Æ6.0
87Æ22
ND
1.9Æ1.6
1.9Æ1.6
[a] Compound concentration required to inhibit tumor cell proliferation by 50%; data are expressed as the
mean ÆSE from the dose–response curves of at least three independent experiments; ND: not determined.
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R. Romagnoli et al.
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group located at C6 (compare the activities of C6 methoxy de-
rivatives 3g and h with those of C7 methoxy congeners 3i
and j). As predicted from the results described above, the 3-
methyl analogues 3h and j were more potent than their un-
substituted counterparts 3g and i. In keeping with the findings
described above, derivative 3h, with a C6 methoxy group, was
two- to fourfold more potent than the 3-unsubstituted ana-
logue 3g and 3–30-fold more active than 3j, with a C7 me-
thoxy group. Starting from 3j, switching the positions of the
amino and methoxy groups, to yield 3 f, resulted in a dramatic
loss of activity. Comparing compounds 3g and 3i further con-
firmed the superior antiproliferative activity of having a C6 me-
thoxy group, as in 3g, as opposed to a C7 methoxy group, as
in 3i.
Table 2. Inhibition of tubulin polymerization and colchicine binding by
compounds 3g, 3h, 3i, 3j, 4a, and CA4.
Compd
IC50 ÆSD [mm][a]
Colch. bind.ÆSD [%][b]
3g
3h
3i
3j
4a
1.4Æ0.01
0.56Æ0.08
3.1Æ0.2
1.6Æ0.1
>40
72Æ0.5
96Æ0.09
56Æ6
49Æ0.5
ND
CA-4 (1a)
1.0Æ0.1
99Æ3
[a] Inhibition of tubulin polymerization; the concentration of tubulin was
10 mm. [b] Inhibition of [3H]colchicine binding; tubulin, colchicine, and
test compound were at 1, 5, and 5 mm, respectively; ND: not determined.
Further SAR information that can be derived from 3g and
3h is that the ortho relationship between the amino and the
methoxy groups plays an essential role in maximal activity, per-
haps mimicking the B ring of aminocombretastatin 1c. In con-
clusion, the introduction of an amino group at C5 of 2-(3’,4’,5’-
trimethoxybenzoyl)benzo[b]furan and a methoxy group at C6
are important for maximal cytotoxicity.
cytostatic than 3i. The most potent compound in this series
was compound 3h, with a tubulin polymerization IC50 value of
0.56 mm. This is in agreement with 3h being the compound
with the greatest antiproliferative activity.
In the colchicine binding studies, compound 3h strongly in-
hibited the binding of [3H]colchicine to tubulin as 96% inhibi-
tion occurred when both this agent and colchicine were at a
concentration of 5 mm. This derivative was as active as CA-4,
which in these experiments inhibited colchicine binding by
99%. These data indicate that 3h strongly binds to the colchi-
cine site on tubulin. These data are consistent with the conclu-
sion that tubulin is the intracellular target of compounds 3g–j.
Once the antiproliferative and antitubulin activities were de-
termined, compounds 3g, 3h, 3j, and 4a were selected for
further evaluation on their effects on human myeloid leukemia
HL-60 and U937 cell-cycle distribution by flow cytometric anal-
ysis. Table 3 shows, for each compound, the percentage of
cells in each phase of the cell cycle, whereas Figure 2 summa-
rizes the fractions of cells in the sub-G1 peak, which represents
Starting with compounds 3a–f, linking the a-bromoacryloyl
moiety to the amino group furnished derivatives 4a–f and led
to a substantial increase in antiproliferative activity in all cases.
However, starting with the methoxy substituted analogues 3h,
i, and j, the introduction of the a-bromoacryloyl moiety to pro-
duce 4g, h, and i, the effects were less dramatic. In fact, 4g
and 4i had less activity than their congeners 3h and 3j,
whereas the conversion of 3i to 4h led to only small increases
in activity in four cell lines. For the unsubstituted a-bromoacry-
loylamido derivatives 4a, b, d, and e, the greatest activity oc-
curred when the a-bromoacryloylamido moiety was located at
C4 or C5, the least at C6 or C7 except that the four com-
pounds had similar activity against HeLa cells. Finally, there are
no differences in antiproliferative activities in all cell lines be-
tween 3h and its glycine hydrochloride prodrug 5.
Table 3. Effect of compounds 3g, 3h, 3j, and 4a on cell-cycle phase dis-
tribution of human myeloid leukemia HL-60 and U937 cells.
To investigate whether the antiproliferative activities of
these compounds were related to an interaction with the mi-
crotubule system, the most active compounds, 3g, 3h, 3i, 3j,
and 4a, were evaluated for their in vitro inhibition of tubulin
polymerization and for their inhibitory effects on the binding
of [3H]colchicine to tubulin (in the former assay, tubulin was at
10 mm; in the latter assay, tubulin was at 1 mm, whereas the
compounds and colchicine were at 5 mm).[19] CA-4 was exam-
ined in simultaneous experiments as a reference compound
(Table 2). The derivative 3h, with an IC50 value of 0.56 mm, ex-
hibited antitubulin activity approximately twofold greater than
that of CA-4 (1.0 mm), whereas 3g and 3j, with IC50 values of
1.4 and-1.6 mm, respectively, were slightly less potent than CA-
4. Compound 4a did not greatly alter tubulin assembly at a
concentration as high as 40 mm. Compound 3i was half as
active as 3j. Thus, it is probable that the mechanism of action
of the most potent a-bromoacryloylamido derivative 4a did
not involve tubulin.
Compd
Conc. [mm]
Cell-cycle phase [%]
G1
Sub-G1
S
G2/M
HL-60[a]
Control
3g
3g
3h
3h
3j
4a
–
0.01
0.1
0.01
0.1
0.1
4.2Æ0.2 45.8Æ0.2
3.7Æ0.3 45.5Æ0.1
27.0Æ6.0 25.9Æ9.7
12.1Æ2.7 35.0Æ0.8
36.9Æ5.6 30.0Æ6.0
6.8Æ0.8 41.9Æ3.2
3.9Æ0.1 43.0Æ1.0
12.7Æ0.8 33.3Æ3.1
12.3Æ2.0 34.8Æ3.0
9.8Æ0.7 20.8Æ3.2
11.9Æ0.3 32.3Æ7.6
10.0Æ4.0 24.0Æ5.0
13.1Æ1.3
33.1Æ7.0
0.1
12.5Æ1.3 36.8Æ1.7
U937[a]
Control
3g
3g
3h
3h
3j
4a
–
0.01
0.1
0.01
0.1
0.1
1.4Æ0.4 47.2Æ2.5
1.6Æ0.7 42.9Æ2.4
20.0Æ1.3 13.8Æ0.3
24.7Æ0.6 26.8Æ4.1
24.5Æ2.3 32.3Æ1.1
10.5Æ0.6 49.6Æ0.9
22.1Æ6.8 26.2Æ11.8 17.2Æ1.6 32.3Æ5.4
23.8Æ0.7 17.2Æ0.7
8.1Æ0.4 44.0Æ1.4
3.0Æ1.3 49.9Æ3.8
14.5Æ1.8 38.3Æ2.0
19.7Æ0.2 28.6Æ1.5
23.5Æ1.5 24.5Æ1.5
0.1
[a] Cells were cultured with the indicated concentrations of test com-
pounds for 24 h, and the cell-cycle phase distribution was determined by
flow cytometry; values represent the mean ÆSE of two independent ex-
periments, with three determinations in each.
The order of inhibitory effects on tubulin assembly was 3h
> CA-4 > 3g > 3j > 3i @ 4a, which was consistent with the
results of the antiproliferative assays, except that 4a was more
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Benzo[b]furan-Based Inhibitors
cells in the G2/M phase, as well as cells in the G1 phase of the
cell cycle.
In the U937 cells, there were concentration-dependent in-
creases in both the apoptotic and G2/M fractions with both 3g
and 3h. These increases were accompanied by decreases in
both the G1 and S fractions. In this cell line, the changes were
those expected for treatment with antitubulin agents.
The detection of a significant increase in sub-G1 peak upon
incubation with compounds 3g and 3h in a concentration-de-
pendent manner in both cell lines, suggested that they exert
their growth inhibiting effect by induction of apoptosis. We
then examined further the apoptotic effects of compounds 3g,
3h, 3j, and 4a in the two cell lines.
As shown in Figure 3A (upper row), using fluorescence mi-
croscopy to analyze the morphological changes of U937 cells,
compound 3h induced the appearance of condensed and
fragmented chromatin characteristic of apoptotic cell death at
various concentrations.[20] Control U937 cells showed normal
features, with the nuclei round and homogeneous. Also com-
pounds 3g, 3j, and 4a induced important morphological
changes characteristic of apoptotic cells, as visualized by
phase-contrast microscopy (lower row).
Degradation of DNA into a specific fragmentation pattern is
a characteristic feature of apoptosis.[21] We also examined
whether these compounds induced chromosomal DNA frag-
mentation, which is considered the end point of the apoptotic
pathway resulting from activation of caspase-activated endo-
nuclease. As we expected, when human HL-60 cells were incu-
bated at the indicated concentrations of compounds 3g, 3h,
Figure 2. A) HL-60 and B) U937 cells were treated with the indicated concen-
trations (in mm) of compounds 3g, 3h, 3j, and 4a for 24 h, and apoptosis
was evaluated by flow cytometry. Values represent the mean ÆSE of three
independent experiments, each performed in triplicate.
apoptotic cells. Cells were cul-
tured for 24 h with each com-
pound at 100 nm, and the two
most active agents (3g and 3h)
were also examined at 10 nm.
These data show that 100 nm 4a
had no effect on cell-cycle distri-
bution, consistent with the con-
clusion above that the com-
pound did not target tubulin.
With 100 nm 3j, only a modest
increase in apoptotic cells was
observed. Compounds 3g and
3h had different cell-cycle ef-
fects in the two cell lines.
In HL-60 cells, 3g caused an
increase in apoptotic cells only
at 100 nm, whereas 3h was ef-
fective at both concentrations.
Neither compound, however,
Figure 3. Morphological changes and apoptosis induction by compounds 3g, 3h, 3j, and 4a on human leukemia
caused the increase in G2/M cells cells. A) Upper row: photomicrographs of representative fields of U937 cells stained with Hoechst 33258 to evalu-
ate nuclear chromatin condensation (apoptosis) after treatment with the indicated concentrations of compound
frequently observed with antitu-
3h for 6 h. Lower row: U937 cells were incubated with medium alone (control) or the indicated compounds;
bulin agents. In fact, the increase
images of cells in culture were obtained with an inverted phase-contrast microscope. B) HL-60 cells were incubat-
in apoptotic cells seemed to
ed with the indicated compounds at the indicated concentrations for 6 h, and genomic DNA was extracted, sepa-
come partially at the expense of rated on an agarose gel, and visualized under UV light by ethidium bromide staining.
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R. Romagnoli et al.
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3j, and 4a, the DNA showed the typical fragmentation pat-
terns formed by internucleosomal hydrolysis of chromatin,
which then led to the appearance of DNA ladders when sepa-
rated by agarose gel electrophoresis (Figure 3B).
As the proteolytic processing of caspases is an important
event in caspase-dependent apoptotic cell death, we evaluated
the effect of 3g, 3h, 3j, and 4a on caspases. To this end, HL-
60 and U937 cells were treated with the compounds, and the
initiator (caspase-8 and -9) and executioner (caspase-3 and -6)
caspases were determined by Western blot analysis (Figure 4).
We found that 3g, 3h, 3j, and 4a stimulated the cleavage of
inactive procaspase-9 to the active 37 kDa fragment, and also,
that concentrations of compounds 3g and 3h as low as
0.3 mm significantly promoted procaspase-8 hydrolysis. As ex-
pected, compounds 3g and 3h significantly promoted the
cleavage of inactive pro-caspases-3 and -6 in both cell lines
(Figure 4). Among the different caspases, caspase-3 is consid-
ered to be the key mediator of apoptosis induced by cytotoxic
Apoptosis is mediated by the activation of a family of cys-
teine aspartyl-specific proteases known as caspases, expressed
as inactive pro-enzymes in living cells that are sequentially acti-
vated by specific proteolytic cleavage.[22] Caspase-8 is the main
executer of the extrinsic pathway initiated at the plasma mem-
brane by activation of cell surface death receptors, whereas
caspase-9 mediates the mitochondrial pathway. Both caspases,
called apical caspases, are usually the first to be activated in
the apoptotic process, and they subsequently activate down-
stream caspase-3, which finally causes apoptosis.[23]
Figure 4. Involvement of caspases in the induction of apoptosis in human leukemia cells. Cells were incubated in the presence of the indicated concentra-
tions of compounds 3g, 3h, 3j, or 4a, and cell lysates were assayed by immunoblotting for the cleavage of pro-caspases-9, -8, -6, and -3, poly(ADP-ribose)
polymerase (PARP), and cytochrome c release. b-Actin was used as a loading control.
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Benzo[b]furan-Based Inhibitors
agents. The executioner caspase-3 is responsible for the phys-
iological (for example, cleavage of the DNA repair enzyme pol-
y(ADP-ribose) polymerase-1, nuclear lamins, and cytoskeleton
proteins) and morphological changes (DNA-strand breaks, nu-
clear membrane damage, and membrane blebbing) that occur
in apoptosis.[23a]
To determine whether the procaspase-3 processing was as-
sociated with an increase in enzymatic activity, we also exam-
ined the induction of cleavage of poly(ADP-ribose) polymerase
(PARP), a known nuclear enzyme substrate of caspase-3 that
plays an important role in DNA repair.[24] Hydrolysis of PARP
guarantees cellular disassembly and supports a role for this
protein as a key regulator of apoptosis in cells treated with
tested compounds.[25] As shown in Figure 4, a concentration-
dependent hydrolysis of the 116 kDa PARP protein to the
85 kDa fragment was detected after treatment with com-
pounds 3g and 3h. Compounds 3j and 4a were less potent
than compounds 3g and 3h in inducing PARP cleavage. As
PARP is a substrate of caspase-3, the appearance of the 85 kDa
fragment coincided with the activation of caspase-3, as shown
by the decreased level of the 36 kDa pro-enzyme and the in-
creased levels of the 20 and 18 kDa cleaved procaspase-3.
Mitochondria play a key role in cell death when their mem-
branes become permeabilized. Outer mitochondrial membrane
permeabilization is regulated by different members of the Bcl-
2 family, which constitutes a critical cellular checkpoint in the
intrinsic apoptotic pathway.[26] Release of cytochrome c from
the mitochondria to the cytosol is a central event in apoptotic
signaling.[27] To evaluate whether apoptosis induced by com-
pounds 3g, 3h, 3j, and 4a on HL-60 and U937 cells involves
cytochrome c release from mitochondria to cytosol, dose-re-
sponse experiments were performed, and cytosolic prepara-
tions were analyzed by immunoblotting. Membranes were
stripped and re-probed with b-actin antibody as a loading con-
trol. We found a significant increase in the amount of cyto-
chrome c in the cytosol in both cell lines (15 kDa band)
(Figure 4).
As caspase processing does not always correlate with activi-
ty, enzymatic activities of caspase-3-like proteases (caspase-3/
7), and of caspase-8 and -9 were also investigated in extracts
of control or compound 3h-treated HL-60 and U937 cells.
Treatment was with 0.1 or 0.3 mm 3h for 24 h. Cell lysates were
assayed for cleavage of the colorimetric tetrapeptide sub-
strates DEVD-pNA, IETD-pNA, and LEHD-pNA as specific sub-
strates for caspase-3/7, caspase-8, and caspase-9, respectively.
As shown in Figure 5, increases in caspase-3, -8, and -9 activi-
ties occurred after 24 h of treatment and with a concentration
as low as 0.1 mm. The enzymatic activities of caspase-3, -8, and
-9 increased in U937 cells in a concentration-dependent
manner, although this effect was less pronounced in HL-60
cells. In summary, compound 3h induced apoptosis in U937
and HL-60 cells through both the mitochondrial and death-re-
ceptor pathways.
Figure 5. Kinetics of caspase-3/7, -8, and -9 activation in response to com-
pound 3h. A) HL-60 and B) U937 cells were treated with the indicated con-
centrations of compound 3h and harvested at 24 h. Cell lysates were as-
sayed for caspase-3/7, -8, and -9 activities using the DEVD-pNA, IETD-pNA,
and LEHD-pNA colorimetric substrates, respectively. Results are expressed as
fold increase in caspase activity relative to control. Values represent the
mean ÆS.E.; this histogram is representative of two independent experi-
ments, each performed in triplicate.
prodrug of compound 3h, corresponding to derivative 5, for
its ability to act as a VDA in an in vivo model. As an alternative
method to using ultrasound and magnetic resonance imaging
(MRI) to monitor blood flow in tumors, we used a new near-in-
frared optical technique called spatial frequency domain imag-
ing (SFDI) to measure tissue oxygen saturation changes result-
ing from vascular disruption after administration of 5.[29] Previ-
ously, this technique had been applied to monitoring tumor
responses to CA-4, which alters the hemodynamic parameters,
such as blood volume, blood oxygenation, and blood flow, of
CA-4 and its analogues in clinical development have been
shown to quickly and selectively shut down the blood flow of
tumors.[28] These compounds are therefore called antivascular
or vascular disrupting agents (VDAs). We tested the glycine
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R. Romagnoli et al.
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tumors but not the surrounding normal tissues.[30] A rapid de-
crease in oxygen saturation is a result of vascular disruption
and stoppage of blood flow in tumors. In this study, we mea-
sured the changes of oxygenated hemoglobin ([OHb]), deoxy-
genated hemoglobin ([RHb]), and total hemoglobin ([HbO2])
continuously by using an SFDI system in rat breast cancer
tumors after administration of compound 5 via a bolus i.p. in-
jection (30 mgkgÀ1). Within 10 min of administration, there
was a rapid decrease in oxygen saturation in tumor tissues
similar to what has been observed with CA-4 treatment (data
not shown), confirming that derivative 5 causes vascular dis-
ruption in vivo (Figure 6).
group at the 5- (3 f), 6- (3g and h), and 7- (3 and j) positions.
Inhibition of cell growth was strongly dependent on the rela-
tive positions of the amino and methoxy moieties and on the
presence of a methyl group at the 3-position of the benzo[b]-
furan skeleton. Thus, for the 5-amino derivatives 3b and 3c,
the introduction of a methoxy substituent at the C6 position
resulted in the most active compounds (3g and 3h) of the
series. The addition of a methoxy group at the C6 position re-
sulted in a 100-fold increase in potency as compared with the
parent compound 3c. For compound 3g, moving the methoxy
group from the C6 to the C7 position, to furnish 3i, resulted in
a tenfold decrease in activity, whereas the differences between
C6 and C7 methoxy groups ranged from 3–30-fold for the 3-
methyl derivative 3h. Comparing the 3-unsubstituted deriva-
tive 3g with the 3-methyl counterpart 3h, it was evident that
introduction of a methyl at the C3 position led to a threefold
increase in antiproliferative activity against all cell lines. For
these latter compounds, the 5-amino-6-methoxy benzene por-
tion of the benzo[b]furan skeleton mimicked the structure of
the B ring of aminocombretastatin 1c. The high potency of
compounds 3g and h allowed us to confirm that the ortho re-
lationship between the 3-amino group and the carbonyl
oxygen of the 2-(3’,4’,5’-trimethoxybenzoyl) moiety, as for 2a,
was not required for activity.
Molecular docking studies were also performed to investi-
gate the possible binding mode of the most active compounds
to tubulin. Results obtained for compound 3h (see figure S1 in
Supporting Information) indicated that the amino group in C5
would establish hydrogen bonds with Thr179 and Ser178 and
that the methoxy group at C6 would form a hydrogen bond
with the side chain of Ser178. Compound 3j bound in a very
similar orientation as 3h, with the trimethoxyphenyl moiety in
contact with Cys241 and forming an extensive network of
nonpolar interactions with the colchicine binding pocket. How-
ever, there was also a significant difference in the position of
the benzo[b]furan ring, which shifted slightly to accommodate
the C7 methoxy group. This movement did not allow the for-
mation of all the hydrogen bonds observed for 3h, although
the amino group was still within a distance compatible with a
hydrogen bond with Thr179. The loss of the other hydrogen
bonds could explain the observed loss in biological activity.
All the unsubstituted a-bromoacryloylamido derivatives 4a,
b, d, and e were 10–100-fold more active than their amino
counterparts, demonstrating that the presence of an a-bro-
moacryloyl moiety significantly enhanced antiproliferative ac-
tivity. With the exception of HeLa cells, shifting the a-bromoa-
cryloylamido function from the C4/5 to the C6/7 position, led
to a decrease in activity. Compounds 3h and 3g were able to
induce apoptosis in HL-60 and U937 human leukemia cells,
with 3h being the most effective derivative. Compound 3h at
a concentration of 100 nm increased the percentage of apop-
totic HL-60 and U937 cells by 9- and 17-fold, respectively. Mor-
phological changes characteristic of apoptotic cells (fragment-
ed and condensed chromatin) were demonstrated by fluores-
cence microscopy. Apoptosis induced by compound 3h was
characterized by the activation of caspases-3, -8, and -9. More-
over, Western blot evaluation of extracts prepared from cells
treated with these compounds demonstrated cleavage of
116 kDa PARP with the generation of the 85 kDa fragment usu-
ally observed in apoptotic cells. The potential glycine prodrug
of compound 3h (compound 5) caused vascular disruption
in vivo and thus represents a new vascular disrupting agent.
Conclusions
In the studies presented herein, we investigated molecules
bearing an amino substituent at the 4-, 5-, 6-, and 7-positions
of the benzene portion of the benzo[b]furan skeleton (com-
pounds 3a–e), with the concomitant presence of a methoxy
Experimental Section
Chemistry
1
General: H and 13C NMR spectra were recorded on Bruker AC 200
and Varian 400 Mercury Plus spectrometers, respectively. Chemical
shifts (d) are given in ppm upfield from tetramethylsilane (TMS) as
internal standard, and the spectra were recorded in appropriate
deuterated solvents, as indicated. Melting points (mp) were deter-
mined on a Bꢂchi–Tottoli apparatus and are uncorrected. All prod-
Figure 6. Compound 5 induced a rapid decrease in oxygen saturation in a
rat breast cancer model. The spatial frequency domain imaging technique
was used to obtain real-time images of tumors noninvasively. Compound 5
was given at the time point indicated by the dashed line, and monitoring
was continued for 1 h. After data were collected and analyzed, the tumor
tissue oxygen saturation (StO2) was plotted over time.
1
ucts reported showed H and 13C NMR spectra in agreement with
the assigned structures. Elemental analyses were conducted by the
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Benzo[b]furan-Based Inhibitors
Microanalytical Laboratory of the Chemistry Department of the
University of Ferrara. Mass spectra were obtained by electrospray
ionization (ESI) in positive mode using an ESI Micromass ZMD 2000
mass spectrometer. All reactions were carried out under an inert
atmosphere of dry N2 gas, unless otherwise described. Standard sy-
ringe techniques were applied for transferring dry solvents. Reac-
tion courses and product mixtures were routinely monitored by
TLC on silica gel (pre-coated F254 Merck plates) and visualized with
aqueous KMnO4. Flash chromatography was performed using 230–
400 mesh silica gel and the indicated solvent system (petroleum
ether (PE), bp: 40–608C, was employed). Organic solutions were
dried over anhydrous Na2SO4. CaCl2 was used in the distillation of
DMF, and the distilled solvent was stored over molecular sieves
(3 ꢃ).
2H), 3.92 (s, 6H), 3.94 (s, 3H), 6.89 (d, J=9.0 Hz, 1H), 6.91 (s, 1H),
7.29 (d, J=9.0 Hz, 1H), 7.34 ppm (s, 2H); 13C NMR (100 MHz,
CDCl3): d=10.5, 56.4, 56.6, 61.2, 102.7, 105.4, 107.6, 107.9, 113.2,
116.6, 129.5, 129.7, 132.6, 142.2, 142.6, 151.5, 153.4, 157.6,
182.6 ppm; MS (ESI): [M+1]+ =343.6; Anal. calcd for C19H19NO5: C
66.85, H 5.61, N 4.10, found: C 66.72, H 5.38, N 4.01.
(6-Aminobenzo[b]furan-2-yl)(3,4,5-trimethoxyphenyl)methanone
(3d): Following general procedure B, the residue was subjected to
chromatography with EtOAc/PE (3:7) as eluent to give 3d as a
yellow solid (239 mg, 73%): Rf =0.34 (PE/EtOAc, 7:3); mp: 173–
1
1758C; H NMR (200 MHz, CDCl3): d=3.94 (s, 6H), 3.95 (s, 3H), 4.04
(bs, 2H), 6.88 (dd, J=8.4, 2.0 Hz, 1H), 6.85 (s, 1H), 7.27 (s, 2H), 7.42
(s, 1H), 7.47 ppm (dd, J=8.4, 2.0 Hz, 1H); 13C NMR (100 MHz,
CDCl3): d=56.1, 56.5, 61.4, 102.3, 105.4, 107.5, 107.9, 113.2, 116.2,
129.7, 130.0, 132.4, 142.2, 142.6, 151.3, 153.2, 157.5, 182.7 ppm; MS
(ESI): [M+1]+ =328.1; Anal. calcd for C18H17NO5: C 66.06, H 5.23, N
4.28, found: C 65.91, H 5.00, N 4.11.
General procedure (A) for the synthesis of nitro-2-(3,4,5-trime-
thoxybenzoyl)benzo[b]furans 7a–j: 2-Bromo-1-(3,4,5-trimethoxy-
phenyl)ethanone (289 mg, 1 mmol) and anhydrous K2CO3 (276 mg,
2 mmol) was added to a solution of the appropriate substituted ni-
trosalicylaldehyde or nitro-2-hydroxyacetophenone (1 mmol) in dry
acetone (15 mL) while stirring, and the reaction mixture was held
at reflux for 18 h. After cooling, the reaction mixture was evaporat-
ed and the residue was dissolved in a mixture of CH2Cl2 (15 mL)
and H2O (5 mL). The organic layer was washed with brine, dried,
and concentrated in vacuo to obtain a residue, which was purified
by crystallization from PE.
(7-Aminobenzo[b]furan-2-yl)(3,4,5-trimethoxyphenyl)methanone
(3e): Following general procedure B, the residue was subjected to
chromatography with EtOAc/PE (3:7) as eluent to give 3e as a
yellow solid (150 mg, 46%): Rf =0.34 (PE/EtOAc, 7:3); mp: 116–
1
118 8C; H NMR (200 MHz, CDCl3): d=3.92 (s, 3H), 3.94 (s, 6H), 7.23
(m, 3H), 7.29 (s, 2H), 7.54 (s, 1H), 8.12 (d, J=8.4 Hz, 1H), 8.42 ppm
(d, J=8.4 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=56.4, 56.8, 61.2,
102.6, 105.7, 107.4, 107.8, 113.4, 116.1, 129.6, 129.9, 132.3, 142.2,
142.5, 151.8, 153.3, 157.4, 182.6 ppm; MS (ESI): [M+1]+ =328.2;
Anal. calcd for C18H17NO5: C 66.06, H 5.23, N 4.28, found: C 65.82, H
5.12, N 4.12.
General procedure (B) for the synthesis of amino 2-(3,4,5-trime-
thoxybenzoyl)benzo[b]furans 3a–j: A mixture of nitro derivative
7a–j (1 mmol) and iron powder (400 mg) was added to a mixture
of EtOH (5 mL), acetic acid (3.5 mL), H2O (2 mL) and 35% HCl
(2 drops). The suspension was held at reflux for 40 min, cooled,
and filtered through Celite. The filtrate was diluted with H2O
(25 mL) and extracted with CH2Cl2 (3ꢄ10 mL). The organic layers
were combined, sequentially washed with 5% aqueous NaHCO3
(10 mL) and H2O (10 mL), dried over Na2SO4, and evaporated. The
residue was purified by flash chromatography on silica gel.
(7-Amino-3-methyl-5-methoxybenzo[b]furan-2-yl)(3,4,5-trime-
thoxyphenyl)methanone (3 f): Following general procedure B, the
residue, purified by column chromatography with EtOAc/PE (4:6)
as eluent, furnished 3 f as a yellow solid (241 mg, 65%): Rf =0.36
1
(PE/EtOAc, 1:1); mp: 193–1948C; H NMR (200 MHz, CDCl3): d=2.57
(s, 3H), 3.84 (s, 6H), 3.92 (s, 3H), 3.95 (s, 3H), 3.98 (bs, 2H), 6.45 (s,
1H), 6.48 (s, 1H), 7.37 ppm (s, 2H); 13C NMR (100 MHz, CDCl3): d=
10.6, 55.9, 56.4, 56.6, 61.1, 91.7, 102.9, 107.4, 107.6, 127.7, 129.8,
132.8, 133.0, 139.5, 142.1, 148.6, 152.9, 153.2, 157.5, 184.5 ppm; MS
(ESI): [M+1]+ =372.4; Anal. calcd for C20H21NO6: C 64.68, H 5.70, N
3.77, found: C 64.57, H 5.56, N 3.69.
(4-Aminobenzo[b]furan-2-yl)(3,4,5-trimethoxyphenyl)methanone
(3a): Following general procedure B, the residue was recrystallized
with PE to give 3a as a yellow solid (235 mg, 72%): Rf =0.34 (PE/
EtOAc, 1:1); mp: 67–698C; 1H NMR (200 MHz, CDCl3): d=3.94 (s,
3H), 3.96 (s, 6H), 4.10 (bs, 2H), 6.56 (d, J=8.4 Hz, 1H), 6.98 (d, J=
8.4 Hz, 1H), 7.16 (s, 1H), 7.35 (s, 2H), 7.42 ppm (t, J=8.4 Hz, 1H);
13C NMR (100 MHz, CDCl3): d=56.5, 56.7, 61.1, 102.4, 105.9, 107.2,
107.7, 113.6, 116.0, 129.7, 129.9, 132.5, 142.0, 142.4, 151.1, 153.1,
157.3, 182.9 ppm; MS (ESI): [M+1]+ =328.2; Anal. calcd for
C18H17NO5: C 66.06, H 5.23, N 4.28, found: C 65.85, H 5.05, N 4.09.
(5-Amino-6-methoxybenzo[b]furan-2-yl)(3,4,5-trimethoxyphe-
nyl)methanone (3g): Following general procedure B, the residue
was subjected to chromatography with EtOAc/PE (1:1) as eluent
and furnished 3g as a yellow solid (221 mg, 62%): Rf =0.34 (PE/
1
EtOAc, 7:3); mp: 138–1398C; H NMR (200 MHz, CDCl3): d=3.94 (s,
6H), 3.95 (s, 3H), 3.96 (s, 3H), 4.04 (bs, 2H), 6.92 (s, 1H), 7.03 (s,
1H), 7.28 (s, 2H), 7.37 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=
56.0, 56.2, 56.5, 61.1, 94.2, 105.1, 105.3, 107.0, 107.2, 116.6, 116.8,
119.9, 133.0, 133.2, 134.8, 150.6, 151.6, 153.1, 182.7 ppm; Anal.
calcd for C19H19NO6: C 63.86, H 5.36, N 3.92, found: C 63.64, H 5.22,
N 3.78.
(5-Aminobenzo[b]furan-2-yl)(3,4,5-trimethoxyphenyl)methanone
(3b): Following general procedure B, the residue was subjected to
chromatography with EtOAc/PE (1:1) as eluent to give 3b as a
yellow solid (255 mg, 78%): Rf =0.38 (PE/EtOAc, 1:1); mp: 108–
110 8C; 1H NMR (200 MHz, CDCl3): d=3.66 (bs, 2H), 3.94 (s, 6H),
3.95 (s, 3H), 6.93 (d, J=8.6 Hz, 1H), 6.94 (s, 1H), 7.33 (s, 2H), 7.39
(s, 1H), 7.42 ppm (d, J=8.6 Hz, 1H); 13C NMR (100 MHz, CDCl3):
d=56.4, 56.7, 61.1, 102.5, 105.8, 107.3, 107.8, 113.4, 116.1, 129.6,
129.8, 132.4, 142.3, 142.6, 151.3, 153.2, 157.4, 182.6 ppm; MS (ESI):
[M+1]+ =328.1; Anal. calcd for C18H17NO5: C 66.06, H 5.23, N 4.28,
found: C 65.92, H 5.02, N 4.11.
(5-Amino-3-methyl-6-methoxybenzo[b]furan-2-yl)(3,4,5-trime-
thoxyphenyl)methanone (3h): Following general procedure B, the
residue was subjected to chromatography with EtOAc/PE (1:1) as
eluent to give 3h as a yellow solid (293 mg, 79%): Rf =0.38 (PE/
EtOAc, 7:3); mp: 135–1368C; 1H NMR (200 MHz, CDCl3): d=1.88
(bs, 2H), 2.56 (s, 3H), 3.93 (s, 6H), 3.94 (s, 6H), 6.90 (s, 1H), 6.92 (s,
1H), 7.37 ppm (s, 2H); 13C NMR (100 MHz, CDCl3): d=10.5, 56.1,
56.2, 56.4, 61.1, 94.0, 103.4, 103.7, 107.1, 107.4, 122.2, 127.6, 133.5,
134.2, 148.0, 149.5, 150.6, 152.8, 153.0, 183.9 ppm; MS (ESI): [M+
1]+ =372.0; Anal. calcd for C20H21NO6: C 64.68, H 5.70, N 3.77,
found: C 64.48, H 5.52, N 3.58.
(5-Amino-3-methylbenzo[b]furan-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (3c): Following general procedure B, the residue was
subjected to chromatography with EtOAc/PE (1:1) as eluent to give
3c as a yellow solid (188 mg, 55%): Rf =0.38 (PE/EtOAc, 1:1); mp:
162–1648C; 1H NMR (200 MHz, CDCl3): d=2.56 (s, 3H), 3.64 (bs,
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1
(5-Amino-7-methoxybenzo[b]furan-2-yl)(3,4,5-trimethoxyphe-
nyl)methanone (3i): Following general procedure B, the residue
was subjected to chromatography with EtOAc/PE (1:1) as eluent to
give 3i as a yellow solid (200 mg, 56%): Rf =0.35 (PE/EtOAc, 7:3);
(PE/EtOAc, 1:1); mp: 99–1008C; H NMR (200 MHz, CDCl3): d=2.62
(s, 3H), 3.93 (s, 6H), 3.96 (s, 3H), 6.18 (d, J=1.6 Hz, 1H), 7.17 (d, J=
1.6 Hz, 1H), 7.40 (s, 2H), 7.52 (s, 2H), 8.14 (s, 1H), 8.51 ppm (bs,
1H); 13C NMR (100 MHz, CDCl3): d=10.3, 29.8, 56.2, 56.4, 61.1,
107.2, 107.5, 112.6, 113.1, 121.8, 122.5, 126.9, 129.0, 129.8, 132.6,
1
mp: 140–1428C; H NMR (200 MHz, CDCl3): d=3.78 (bs, 2H), 3.94
(s, 3H), 3.95 (s, 6H), 3.98 (s, 3H), 6.40 (d, J=2.2 Hz, 1H), 6.53 (d, J=
2.2 Hz, 1H), 7.40 (s, 1H), 7.42 ppm (s, 2H); 13C NMR (100 MHz,
CDCl3): d=56.1, 56.4, 56.6, 61.1, 97.9, 100.3, 100.8, 107.3, 107.4,
115.5, 115.9, 129.2, 132.2, 133.8, 144.0, 146.4, 151.1, 153.0,
182.8 ppm; MS (ESI): [M+1]+ =358.4; Anal. calcd for C19H19NO6: C
63.86, H 5.36, N 3.92, found: C 63.73, H 5.20, N 3.81.
132.9, 142.5, 149.3, 151.6, 153.0, 159.1, 184.5 ppm; MS (ESI): [M]+
=
474.5, [M+2]+ =476.5; Anal. calcd for C22H20BrNO6: C 55.71, H
4.25, N 2.95, found: C 55.58, H 4.11, N 2.77.
2-Bromo-N-[2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]furan-6-yl]a-
crylamide (4d): Following general procedure C, the crude residue
purified by flash chromatography using EtOAc/PE (4:6 v/v) as
eluent furnished 4d as a white solid (212 mg, 46%): Rf =0.38 (PE/
(5-Amino-3-methyl-7-methoxybenzo[b]furan-2-yl)(3,4,5-trime-
thoxyphenyl)methanone (3j): Following general procedure B, the
residue, purified by column chromatography with EtOAc/PE (7:3)
as eluent, furnished 3j as a yellow solid (156 mg, 42%): Rf =0.34
1
EtOAc, 1:1); mp: 159–1608C; H NMR (200 MHz, CDCl3): d=3.95 (s,
6H), 3.96 (s, 3H), 6.18 (d, J=1.6 Hz, 1H), 7.17 (d, J=1.6 Hz, 1H),
7.24 (s, 2H), 7.33 (d, J=8.8 Hz, 1H), 7.55 (s, 1H), 7.74 (d, J=8.8 Hz,
1H), 8.26 (s, 1H), 8.61 ppm (bs, 1H); 13C NMR (100 MHz, CDCl3):
d=29.4, 56.2, 56.5, 61.1, 107.2, 107.3, 115.9, 116.1, 117.2, 122.3,
123.6, 124.1, 129.3, 132.1, 137.4, 142.6, 153.1, 153.3, 156.4, 158.9,
182.7 ppm; MS (ESI): [M]+ =460.4, [M+2]+ =462.4; Anal. calcd for
C21H18BrNO6: C 54.80, H 3.94, N 3.04, found: C 54.67, H 3.77, N 2.93.
1
(PE/EtOAc, 7:3); mp: 179–1808C; H NMR (200 MHz, CDCl3): d=2.57
(s, 3H), 3.76 (bs, 2H), 3.93 (s, 6H), 3.95 (s, 6H), 6.39 (d, J=1.0 Hz,
1H), 6.49 (d, J=1.0 Hz, 1H), 7.50 ppm (s, 2H); 13C NMR (100 MHz,
CDCl3): d=10.4, 56.2, 56.3, 56.5, 61.0, 96.5, 96.7, 100.8, 107.4, 107.6,
126.8, 131.4, 132.8, 138.8, 142.1, 143.6, 146.3, 148.9, 152.9,
187.6 ppm; [M+1]+ =372.4; Anal. calcd for C20H21NO6: C 64.68, H
5.70, N 3.77, found: C 64.51, H 5.53, N 3.61.
2-Bromo-N-[2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]furan-7-yl]a-
crylamide (4e): Following general procedure C, the crude residue
purified by flash chromatography using EtOAc/PE (3:7 v/v) as
eluent furnished 4e as a brown solid (290 mg, 63%): Rf =0.34 (PE/
General Procedure (C) for the synthesis of compounds 4a–i:
EDCI (383 mg, 2 mmol) and HOBt (270 mg, 2 mmol) were added to
an ice-cooled solution of aminobenzo[b]furan (1 mmol) in dry DMF
(5 mL), followed by a-bromoacrylic acid (2 mmol, 306 mg). The re-
action was stirred at RT for 18 h and then concentrated in vacuo.
The residue was dissolved with a mixture of CH2Cl2 (15 mL) and
H2O (5 mL), and the organic phase was washed with brine (5 mL),
dried over Na2SO4, and evaporated to dryness in vacuo. The result-
ing crude residue was purified by flash chromatography on silica
gel.
1
EtOAc, 7:3); mp: 184–1858C; H NMR (200 MHz, CDCl3): d=3.94 (s,
6H), 3.96 (s, 3H), 6.20 (d, J=1.8 Hz, 1H), 7.16 (d, J=1.8 Hz, 1H),
7.33 (s, 2H), 7.39 (t, J=8.0 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H), 7.58 (s,
1H), 8.35 (d, J=8.0 Hz, 1H), 8.97 ppm (bs, 1H); 13C NMR (100 MHz,
CDCl3): d=29.8, 56.2, 56.6, 61.2, 107.1, 107.3, 116.1, 116.4, 118.9,
122.2, 123.2, 124.9, 127.4, 129.3, 132.0, 142.9, 146.5, 152.5, 153.2,
159.2, 182.8 ppm; MS (ESI): [M]+ =460.3, [M+2]+ =462.3; Anal.
calcd for C21H18BrNO6: C 54.80, H 3.94, N 3.04, found: C 54.60, H
3.72, N 2.91.
2-Bromo-N-[2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]furan-4-yl]a-
crylamide (4a): Following general procedure C, the crude residue
purified by flash chromatography using EtOAc/PE (3:7 v/v) as
eluent furnished 4a as a white solid (202 mg, 44%): Rf =0.36 (PE/
2-Bromo-N-[3-methyl-5-methoxy-2-(3,4,5-trimethoxybenzoyl)-1-
benzo[b]furan-7-yl]acrylamide (4 f): Following general procedur-
e C, the crude residue purified by flash chromatography using
EtOAc/PE (4:6 v/v) as eluent furnished 4 f as a white solid (262 mg,
1
EtOAc, 7:3); mp: 152–1548C; H NMR (200 MHz, CDCl3): d=3.95 (s,
1
6H), 3.97 (s, 3H), 6.21 (d, J=1.8 Hz, 1H), 7.18 (d, J=1.8 Hz, 1H),
7.39 (s, 2H), 7.43 (t, J=8.8 Hz, 1H), 7.49 (s, 1H), 7.60 (s, 1H), 7.72
(d, J=8.8 Hz, 1H), 8.74 ppm (bs, 1H); 13C NMR (100 MHz, CDCl3):
d=29.9, 56.2, 56.4, 61.1, 107.1, 107.3, 110.0, 110.2, 113.9, 115.9,
120.4, 122.1, 128.9, 129.6, 131.4, 131.9, 151.8, 153.2, 156.4, 158.8,
182.9 ppm; MS (ESI): [M]+ =460.4, [M+2]+ =462.6; Anal. calcd for
C21H18BrNO6: C 54.80, H 3.94, N 3.04, found: C 54.62, H 3.68, N 2.88.
52%): Rf =0.38 (PE/EtOAc, 1:1); mp: 162–1638C; H NMR (200 MHz,
CDCl3): d=2.63 (s, 3H), 3.90 (s, 3H), 3.93 (s, 6H), 3.95 (s, 3H), 6.18
(d, J=1.8 Hz, 1H), 6.88 (d, J=1.8 Hz, 1H), 7.16 (d, J=2.2 Hz, 1H),
7.36 (s, 2H), 8.04 (d, J=2.2 Hz, 1H), 8.82 ppm (bs, 1H); 13C NMR
(100 MHz, CDCl3): d=10.3, 29.7, 56.2, 56.3, 56.6, 61.1, 99.2, 107.2,
107.5, 108.5, 121.9, 123.2, 127.6, 129.6, 129.7, 132.8, 140.0, 142.5,
148.8, 153.0, 156.9, 158.8, 183.2 ppm; MS (ESI): [M]+ =504.2, [M+
2]+ =506.2; Anal. calcd for C23H22BrNO7: C 54.78, H 4.40, N 2.78,
found: C 54.67, H 4.26, N 2.56.
2-Bromo-N-[2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]furan-5-yl]a-
crylamide (4b): Following general procedure C, the crude residue
purified by flash chromatography using EtOAc/PE (1:1 v/v) as
eluent furnished 4b as a brown solid (235 mg, 51%): Rf =0.38 (PE/
2-Bromo-N-[3-methyl-6-methoxy-2-(3,4,5-trimethoxybenzoyl)-1-
benzo[b]furan-5-yl]acrylamide (4g): Following general procedur-
e C, the crude residue purified by flash chromatography using
EtOAc/PE (4:6 v/v) as eluent furnished 4g as a white solid (267 mg,
1
EtOAc, 1:1); mp: 165–1668C; H NMR (200 MHz, CDCl3): d=3.95 (s,
6H), 3.97 (s, 3H), 6.18 (d, J=1.6 Hz, 1H), 7.17 (d, J=1.6 Hz, 1H),
7.33 (s, 2H), 7.58 (dd, J=8.8, 2.2 Hz, 1H), 7.61 (d, J=8.8 Hz, 1H),
8.22 (d, J=2.2 Hz, 1H), 8.26 (s, 1H), 8.52 ppm (bs, 1H); 13C NMR
(100 MHz, CDCl3): d=29.6, 56.4, 56.6, 61.1, 107.2, 107.4, 113.0,
113.2, 114.7, 116.1, 117.3, 121.9, 122.5, 127.5, 129.0, 132.1, 133.3,
1
53%): Rf =0.38 (PE/EtOAc, 1:1); mp: 179–1808C; H NMR (200 MHz,
CDCl3): d=2.60 (s, 3H), 3.93 (s, 3H), 3.96 (s, 3H), 4.02 (s, 6H), 6.16
(d, J=1.6 Hz, 1H), 7.04 (s, 1H), 7.14 (d, J=1.6 Hz, 1H), 7.36 (s, 2H),
8.74 (s, 1H), 9.24 ppm (bs, 1H); 13C NMR (100 MHz, CDCl3): d=
10.5, 29.8, 56.2, 56.4, 56.7, 61.1, 107.1, 107.4, 111.3, 122.2, 123.0,
124.7, 128.3, 128.5, 133.2, 140.0, 142.1, 148.5, 150.8, 151.7, 152.9,
158.7, 184.0 ppm; MS (ESI): [M]+ =504.5, [M+2]+ =506.5; Anal.
calcd for C23H22BrNO7: C 54.78, H 4.40, N 2.78, found: C 54.62, H
4.21, N 2.58.
153.2, 153.3, 159.0, 183.1 ppm; MS (ESI): [M]+ =460.4, [M+2]+
=
462.4; Anal. calcd for C21H18BrNO6: C 54.80, H 3.94, N 3.04, found: C
54.70, H 3.91, N 2.91.
2-Bromo-N-[3-methyl-2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]fur-
an-5-yl]acrylamide (4c): Following general procedure C, the crude
residue purified by flash chromatography using EtOAc/PE (4:6 v/v)
as eluent furnished 4c as a white solid (313 mg, 66%): Rf =0.35
2-Bromo-N-[7-methoxy-2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]-
furan-5-yl]acrylamide (4h): Following general procedure C, the
1850
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1841 – 1853

Benzo[b]furan-Based Inhibitors
crude residue purified by flash chromatography using EtOAc/PE
(6:4 v/v) as eluent furnished 4h as a yellow solid (255 mg, 52%):
Rf =0.33 (PE/EtOAc, 1:1); mp: 170–1728C; 1H NMR (200 MHz,
CDCl3): d=3.95 (s, 6H), 3.97 (s, 3H), 4.04 (s, 3H), 6.17 (d, J=1.8 Hz,
1H), 7.16 (d, J=1.8 Hz, 1H), 7.42 (s, 2H), 7.53 (s, 1H), 7.55 (s, 1H),
7.56 (s, 1H), 8.45 ppm (bs, 1H); 13C NMR (100 MHz, CDCl3): d=
29.8, 56.2, 56.4, 56.6, 61.1, 103.8, 106.0, 107.2, 107.4, 115.3, 115.8,
128.4, 129.0, 131.9, 134.0, 146.1, 149.2, 153.1, 153.6, 159.0, 161.2,
182.4 ppm; MS (ESI): [M]+ =490.2, [M+2]+ =492.2; Anal. calcd for
C22H20BrNO7: C 53.89, H 4.11, N 2.86, found: C 53.69, H 3.88, N 2.65.
aliquots were frozen at À208C. Poly(vinylidene difluoride) (PVDF)
membranes were purchased from Millipore (Billerica, MA, USA). Ac-
rylamide, bisacrylamide, ammonium persulfate, and N,N,N’,N’-tetra-
methylethylenediamine were from Bio-Rad (Hercules, CA, USA). An-
tibodies for poly(ADP-ribose) polymerase (PARP) and caspase-3
were purchased from Stressgen (Victoria, BC, Canada). An antibody
against cytochrome c was purchased from BD PharMingen (San
Diego, CA, USA). Anti-caspase-6 monoclonal antibody and antibod-
ies against caspase-8 and caspase-9 were from Medical & Biological
Laboratories (Nagoya, Japan). Secondary antibodies were from GE
Healthcare (Little Chalfont, UK). All other chemicals were obtained
from Sigma (St. Louis, MO, USA).
2-Bromo-N-[3-methyl-7-methoxy-2-(3,4,5-trimethoxybenzoyl)-1-
benzo[b]furan-5-yl]acrylamide (4i): Following general procedur-
e C, the crude residue purified by flash chromatography using
EtOAc/PE (4:6 v/v) as eluent furnished 4i as a white solid (262 mg,
Cell growth inhibitory activity: Murine leukemia L1210, murine
mammary carcinoma FM3A, human T-lymphocyte Molt/4 and CEM,
and human cervix carcinoma (HeLa) cells were suspended at
300000–500000 cellsmLÀ1 of culture medium, and 100 mL cell sus-
pension was added to 100 mL of an appropriate dilution of the test
compounds in wells of 96-well microtiter plates. After incubation
at 378C for two days, cell number was determined using a Coulter
counter. The IC50 value was defined as the compound concentra-
tion required to inhibit cell proliferation by 50%.
1
52%): Rf =0.34 (PE/EtOAc, 4:6); mp: 162–1638C; H NMR (200 MHz,
CDCl3): d=2.62 (s, 3H), 3.94 (s, 6H), 3.96 (s, 3H), 4.02 (s, 3H), 6.18
(d, J=1.6 Hz, 1H), 7.18 (d, J=1.6 Hz, 1H), 7.49 (s, 1H), 7.51 (s, 2H),
7.56 (s, 1H), 8.51 ppm (bs, 1H); 13C NMR (100 MHz, CDCl3): d=
10.4, 29.6, 56.1, 56.2, 56.6, 61.3, 107.1, 107.3, 111.2, 122.0, 123.2,
124.5, 128.1, 128.6, 133.3, 140.2, 142.3, 148.5, 150.6, 151.5, 152.7,
158.6, 182.4 ppm; MS (ESI): [M]+ =504.2, [M+2]+ =506.2; Anal.
calcd for C23H22BrNO7: C 54.78, H 4.40, N 2.78, found: C 54.59, H
4.20, N 2.61.
HL-60 and U937 cells were cultured in RPMI 1640 medium contain-
ing 10% (v/v) heat-inactivated fetal bovine serum, 100 unitsmLÀ1
penicillin and 100 mgmLÀ1 streptomycin at 378C in a humidified at-
mosphere containing 5% CO2. Cell numbers was determined by
counting in a hematocytometer, and cell viability was always
>95% in all experiments as assayed by the trypan blue exclusion
method. Further dilutions of stock solutions of compounds were
made in culture media just before use. In all experiments, the final
concentration of DMSO did not exceed 0.1% (v/v), a concentration
which is nontoxic to the cells. The same concentration was present
in control cultures.
tert-Butyl-(2-{[6-methoxy-3-methyl-2-(3,4,5-trimethoxybenzoyl)-
1-benzo[b]furan-5-yl]amino}-2-oxoethyl)carbamate (8): EDCI
(192 mg, 1 mmol) and HOBt (135 mg, 1 mmol) were added to an
ice-cooled solution of 3h (186 mg, 0.5 mmol) in dry DMF (5 mL),
followed by the addition of N-(tert-butoxycarbonyl)glycine
(0.6 mmol, 105 mg). The reaction was stirred at RT for 18 h and
then concentrated in vacuo. The residue was dissolved with a mix-
ture of CH2Cl2 (10 mL) and H2O (4 mL), and the organic phase was
washed with brine (5 mL), dried over Na2SO4, and evaporated to
dryness in vacuo. The resulting crude residue was purified by flash
chromatography on silica gel with EtOAc/PE (6:4 v/v) as eluent to
furnish 8 as a yellow powder (438 mg, 83%): Rf =0.36 (PE/EtOAc,
Effects on tubulin polymerization and on colchicine binding to tubu-
lin: Bovine brain tubulin was purified as described previously.[31] To
evaluate the effect of the compounds on tubulin assembly in vitro,
varying concentrations were pre-incubated with 10 mm tubulin in
glutamate buffer at 308C and then cooled to 08C. After addition of
GTP, the mixtures were transferred to 08C cuvettes in a recording
spectrophotometer and warmed to 308C, and the assembly of tu-
bulin 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 com-
pounds to inhibit colchicine binding to tubulin was measured as
described,[19b] except that the reaction mixtures contained 1 mm tu-
bulin, 5 mm [3H]colchicine, and 5 mm test compound.
1
7:3); mp: 90–918C; H NMR (200 MHz, CDCl3) d=1.49 (s, 8H), 1.99
(m, 2H), 2.59 (s, 3H), 3.93 (s, 6H), 3.95 (s, 3H), 3.96 (s, 3H), 5.22 (bs,
1H), 7.00 (s, 1H), 7.34 (s, 2H), 8.51 (bs, 1H), 8.67 ppm (s, 1H);
13C NMR (100 MHz, CDCl3): d=10.4, 28.4 (3C), 45.8, 56.0, 56.4, 56.6,
61.1, 83.4, 95.7, 107.1, 107.4, 108.5, 113.4, 113.6, 120.8, 124.3, 127.0,
132.7, 140.2, 143.9, 153.1, 154.1, 154.6, 168.2, 188.0 ppm; MS (ESI):
[M+1]+ =529.7.
N-[6-methoxy-3-methyl-2-(3,4,5-trimethoxybenzoyl)-1-benzo[b]-
furan-5-yl]glycinamide hydrochloride (5): The N-Boc-glycine de-
rivative 8 (132 mg, 0.25 mmol) was added to a saturated solution
of HCl in MeOH (10 mL). The mixture was stirred for 3 h at RT and
evaporated at reduced pressure. The residue was suspended in
Et2O (5 mL), and the mixture was stirred for 1 h and filtered to fur-
nish the final compound as a grey solid (460 mg, >95%): Rf =0.28
Evaluation of apoptosis: The rate of apoptotic cell death was ana-
lyzed by fluorescence microscopy and by flow cytometric analysis
of propidium iodide (PI)-stained nuclei as described below.
1
(CH2Cl2/MeOH, 4:6); mp: 250–2518C; H NMR (200 MHz, [D6]DMSO)
Fluorescence microscopy analysis: Cells were harvested and fixed in
3% paraformaldehyde and incubated at RT for 10 min. The fixative
was removed, and the cells were washed with phosphate-buffered
saline (PBS), resuspended in 30–50 mL of PBS containing
20 mgmLÀ1 bis-benzimide trihydrochloride (Hoechst 33258), and in-
cubated at RT for 15 min. Stained nuclei were visualized using a
Zeiss fluorescence microscope.
d=2.58 (s, 3H), 3.74 (m, 1H), 3.78 (s, 6H), 3.87 (s, 3H), 3.96 (s, 3H),
7.32 (s, 2H), 7.53 (s, 1H), 8.19 (bs, 4H), 8.30 (s, 1H), 9.90 ppm (s,
1H); 13C NMR (100 MHz, CDCl3): d=10.0, 41.0, 56.1, 56.2, 56.6, 60.2,
95.5, 107.0, 107.3, 113.4, 113.6, 120.8, 124.3, 127.0, 132.7, 141.4,
147.6, 151.7, 152.4, 152.6, 165.4, 183.0 ppm; MS (ESI): [M+1]+
=
428.8; Anal. calcd for C22H25ClN2O7: C 56.84, H 5.42, N 6.03, found:
C 56.61, H 5.21, N 5.89.
Quantification of apoptosis by flow cytometry: To study changes in
cell DNA content, histogram measurements of hypodiploid DNA
formation was performed by flow cytometry using a Coulter EP-
ICSTM cytometer (Beckman Coulter). Histograms were analyzed with
the Expo 32 ADC SoftwareTM. Cells were collected and centrifuged
Biology
Reagents: Stock solutions of the various compounds at 100 mm
were made by dissolving them in dimethyl sulfoxide (DMSO), and
ChemMedChem 2011, 6, 1841 – 1853
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1851

R. Romagnoli et al.
MED
at 500 g, washed with PBS, and resuspended in 50 mL of PBS. Fol-
lowing dropwise addition of 1 mL of ice-cold 75% EtOH, fixed cells
were stored at À208C for 1 h. Samples were then centrifuged at
500 g and washed with PBS before resuspension in 1 mL of PBS
containing 50 mgmLÀ1 propidium iodide and 100 mgmLÀ1 RNase A.
The cells were then incubated for 1 h at 378C in the dark. The per-
centage of cells with decreased DNA staining, composed of apop-
totic cells resulting from either fragmentation or decreased chro-
matin, was determined on a minimum of 10000 cells per experi-
mental condition. Cell debris was excluded from analysis by selec-
tive gating based on anterior and right angle scattering.
gauge needle and the lysate spun down at 1000 g for 5 min at
48C to eliminate nuclei and unbroken cells. The supernatant frac-
tion was centrifuged at 15000 g for 20 min at 48C, and the result-
ing pellet was designated as the mitochondrial fraction. The super-
natant was further centrifuged at 105000 g for 45 min at 48C and
the resulting supernatant was designated as the cytosolic fraction.
Cytosolic proteins (50 mg) were resolved on a 15% sodium dodecyl
sulfate/polyacrylamide gel, and cytochrome c was detected by
chemiluminescence as described above.
Assay of caspase activity: After treatments cells were harvested by
centrifugation at 1000 g for 5 min at 48C and washed with PBS,
and the cell pellets were kept on ice. The cells were resuspended
in cell lysis buffer (50 mm HEPES, pH 7.4, 1 mm dithiothreitol,
0.1 mm EDTA, 0.1% Chaps) and held on ice for 5 min. After centri-
fugation for 10 min at 17000 g at 48C, the supernatants were ana-
lyzed for protein concentration by the Bradford assay and stored
at À208C until used to study caspase colorimetric enzymatic activi-
ty. Equal amounts of protein (ꢀ20 mg) from different treatments
were used, and the assays were set up on ice. The net increase of
absorbance at 405 nm after incubation at 378C was indicative of
enzyme activity. Specific labeled substrates for caspase-3, -8, and 9
activities were N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline (DEVD-pNA),
N-acetyl-Ile-Glu-Thr-Asp-p-nitroaniline (IETD-pNA), and N-acetyl-
Leu-Glu-His-Asp-p-nitroaniline (LEHD-pNA) respectively.
Analysis of DNA fragmentation: A late biochemical hallmark of
apoptosis is the fragmentation of the genomic DNA. It is an irrever-
sible event and occurs before changes in plasma membrane per-
meability. DNA isolation and gel electrophoresis were performed as
described previously.[32] Briefly, cells (1ꢄ105) were collected by cen-
trifugation, washed with PBS, and incubated in 30 mL of lysis buffer
[50 mm Tris-HCl (pH 8.0), 10 mm EDTA, 0.5% sodium dodecyl sul-
fate], containing 1 mgmLÀ1 RNase A at 378C for 1 h. Then, 3 mL of
proteinase K (10 mgmLÀ1) was added, and the mixture was incubat-
ed at 508C for an additional 2 h. DNA was extracted with 100 mL of
phenol-chloroform-isoamyl alcohol (24:24:1) and mixed with 5 mL
of loading solution [10 mm EDTA, 1% (w/v) low melting-point agar-
ose, 0.25% bromophenol blue, and 40% sucrose, pH 8.0]. Samples
were separated by electrophoresis in 2% agarose gels in TAE
buffer [40 mm Tris-acetate (pH 8.0), 1 mm EDTA], visualized by ul-
traviolet illumination after ethidium bromide (0.5 mgmLÀ1) staining,
and the images were captured by a digital camera (Digi Doc
system, Bio-Rad).
Spatial frequency domain imaging to measure tissue oxygen satura-
tion change as a result of vascular disruption: To monitor the dy-
namic physiological process associated with treatment in vivo, we
used a fast, noncontact, near-infrared optical technique called spa-
tial frequency domain imaging (SFDI). SFDI projects broadband
light in sinusoidal patterns onto a tissue sample. The backscattered
reflectance is then filtered by wavelength and used to analyze the
optical content. A Monte Carlo forward model is then used to
create a look up table for fitting these optical properties.[34] For this
study, we projected the two spatial frequency patterns of NIR light
with wavelengths from 700 to 850 nm at every 50 nm on the sur-
face of breasts in a rat breast cancer model. After 10 min of base-
line measurement, compound 5 was administered via a bolus i.p.
injection (0.45 mL, 30 mgkgÀ1) and we acquired images for another
60 min to monitor the hemodynamic effects of compound 5 on
tumor and normal breast tissue. Once the images at several wave-
lengths were obtained, quantitative fits for the concentration of
oxy- ([OHb]), deoxy- ([RHb]), total ([THb]) hemoglobin, tissue
oxygen saturation (% StO2 =[OHb]/[THb]ꢄ100), and light scattering
were carried out. The study was approved by the Institutional
Animal Ethical Committee of the Department of Pathology, VA
Medical Center, Long Beach, CA (USA).
Western blot analysis: Cells (1ꢄ106 mL) were treated in the absence
or presence of the indicated concentrations of compounds 3g, 3h,
3j, and 4a for 24 h and harvested by centrifugation at 500 g for
10 min. Cell pellets were resuspended in lysis buffer [20 mm Tris-
HCl (pH 7.4), 2 mm EDTA, 137 mm NaCl, 10% glycerol, 1% Triton X-
100, 2 mm tetrasodium pyrophosphate, 20 mm sodium b-glycero-
phosphate, 10 mm sodium fluoride, 2 mm sodium orthovanadate],
supplemented with protease inhibitors phenylmethylsulfonyl fluo-
ride (PMSF, 1 mm), leupeptin, aprotinin, and pepstatin A (5 mgmLÀ1
each) for 15 min at 48C. The cells were sonicated on ice (five times
for 5 s at 40 W, with 20 s intervals between each sonication) with a
Braun Labsonic 2000 microtip sonifier (Braun, Melsungen, Germa-
ny) and centrifuged at 11000 g for 10 min at 48C. Protein concen-
tration of supernatants was measured by the Bradford method,[33]
and samples containing equal amounts of proteins were boiled in
sodium dodecyl sulfate sample buffer for 5 min before loading on
an sodium dodecyl sulfate-polyacrylamide gel (7.5% for PARP and
12.5% for caspases). Proteins were electrotransfered to PVDF mem-
branes, blocked with 5% fat-free dry milk in Tris-buffered saline
[50 mm Tris-HCl (pH 7.4), 150 mm NaCl] with 0.1% Tween 20, and
then incubated with specific primary antibodies overnight at 48C.
After washing and incubation with an appropriate horseradish per-
oxidase-conjugated secondary antibody, the antigen-antibody
complexes were visualized by enhanced chemiluminescence (Milli-
pore) using the manufacturer’s protocol.
Molecular modeling: All molecular modeling studies were per-
formed on a MacPro dual 2.66 GHz Xeon running Ubuntu 10. The
tubulin structure was downloaded from the Protein Data Bank
(http://www.rcsb.org/; PDB ID: 1SA0).[35] Hydrogen atoms were
added to the protein, using Molecular Operating Environment
(MOE),[36] and minimized keeping all heavy atoms fixed until a
RMSD gradient of 0.05 kcalmolÀ1 ꢃÀ1 was reached. Ligand struc-
tures were built with MOE and minimized using the MMFF94x
Detection of cytochrome c: Release of cytochrome c from mitochon-
dria was detected by Western blot analysis. After treatment, cells
were washed twice with PBS and then resuspended in ice-cold
buffer [20 mm HEPES (pH 7.5), 1.5 mm MgCl2, 10 mm KCl, 1 mm
EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsul-
fonyl fluoride and 5 mgmLÀ1 each of leupeptin, aprotinin, and pep-
statin A] containing 250 mm sucrose. After a 15 min incubation on
ice, cells were lysed by pushing them several times through a 22-
force field until
a
RMSD gradient of 0.05 kcalmolÀ1 ꢃÀ1 was
reached. The docking simulations were performed using
PLANTS.[37]
1852
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1841 – 1853

Benzo[b]furan-Based Inhibitors
available and were used as received; 2-hydroxy-6-nitrobenzaldehyde
(6a) was prepared by following the procedure reported here: L. P. Mir-
anda, W. D. F. Meutermans, M. L. Smythe, P. F. Alewood, J. Org. Chem.
2000, 65, 5460–5468. For the preparation of 6d, see: K. S. MacMillan, T.
Nguyen, I. Hwang, D. L. Boger, J. Am. Chem. Soc. 2009, 131, 1187–1194.
For the preparation of 1-(2-hydroxy-5-methoxy-3-nitrophenyl)ethanone
(6 f), see M. Cushman, H. Zhu, R. L. Geahlen, A. J. Kraker, J. Med. Chem.
1994, 37, 3353–3362. For the synthesis of 2-hydroxy-4-methoxy-5-nitro-
benzaldehyde (6g), see: M. Guru, S. Rao, C. Srikantia, M. S. Iyengar, J.
Chem. Soc. 1924, 556–560. For the preparation of 1-(2-hydroxy-4-me-
thoxy-5-nitrophenyl)ethanone (6h), see: H. Banks, J. Am. Chem. Soc.
1938, 60, 1370. For the synthesis of 2-hydroxy-3-methoxy-5-nitrobenzal-
Acknowledgements
This work was supported by a grant from the Ministry of Science
and Innovation of Spain, from the European Regional Develop-
ment Fund (SAF2010-21380 to F.E.), and the Instituto Canario de
Investigaciꢃn del Cꢂncer (RED PRODNAT CANCER to J.B.). S.E.
thanks the Ministry of Education of Spain for financial support.
The authors thank Marꢁa Teresa Marrero, Dr. Alberto Casolari, Dr.
Erika Marzola, and Dr. Elisa Durini for excellent technical assis-
tance.
´
dehyde (6i), see: M. Kowalewska, H. Kwiecien, Tetrahedron 2008, 64,
5085–5090. For the synthesis of 1-(2-hydroxy-3-methoxy-5-nitropheny-
l)ethanone (6j), see: W. Horton, J. T. Spence, J. Am. Chem. Soc. 1958, 80,
2453–2456.
Keywords: antiproliferative agents
activation · colchicine binding site · tubulin
· apoptosis · caspase
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Received: June 3, 2011
[18] 2-Hydroxy-3-nitrobenzaldehyde (6e), 2-hydroxy-5-nitrobenzaldehyde
(6b), and 1-(2-hydroxy-5-nitrophenyl)ethanone (6c) are commercially
Published online on August 1, 2011
ChemMedChem 2011, 6, 1841 – 1853
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1853