Synthesis and biological evaluation of 2-amino-3-(3′,4′, 5′-trimethoxybenzoyl)-5-aryl thiophenes as a new class of potent antitubulin agents

Article abstract of DOI:10.1021/jm060355e

A new series of compounds in which the 2-amino-5-chlorophenyl ring of phenstatin analogue 7 was replaced with a 2-amino-5-aryl thiophene was synthesized and evaluated for antiproliferative activity and for inhibition of tubulin polymerization and colchicine binding to tubulin. 2-Amino-3-(3′, 4′,5′-trimethoxybenzoyl)-5-phenyl thiophene (9f) as well as the p-fluoro-, p-methyl-, and p-methoxyphenyl substituted analogues (9i, j, and I, respectively) displayed high antiproliferative activities with IC₅₀ values from 2.5 to 6.5 nM against the L1210 and K562 cell lines. Compounds 9i and j were more active than combretastatin A-4 as inhibitors of tubulin polymerization. Molecular docking simulations to the colchicine site of tubulin were performed to determine the possible binding mode of 9i. The results obtained demonstrated that antiproliferative activity correlated well with the inhibition of tubulin polymerization and the lengthening of the G2/M phase of the cell cycle. Moreover, a good correlation was found between these inhibitory effects and the induction of apoptosis in cells treated with the compounds.

Full text of DOI:10.1021/jm060355e

3906
J. Med. Chem. 2006, 49, 3906-3915
Synthesis and Biological Evaluation of 2-Amino-3-(3′,4′,5′-trimethoxybenzoyl)-5-aryl Thiophenes
as a New Class of Potent Antitubulin Agents
Romeo Romagnoli,*,† Pier Giovanni Baraldi,*,† Maria Giovanna Pavani,† Mojgan Aghazadeh Tabrizi,† Delia Preti,†
Francesca Fruttarolo,† Laura Piccagli,† M. Katherine Jung,‡ Ernest Hamel,§ Monica Borgatti,| and Roberto Gambari|
Dipartimento di Scienze Farmaceutiche, UniVersita` di Ferrara, 44100 Ferrara, Italy, SAIC-Frederick Inc., National Cancer Institute at
Frederick, National Institutes of Health, Frederick, Maryland 21702, Toxicology and Pharmacology Branch, DeVelopmental Therapeutics
Program, DiVision of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health,
Frederick, Maryland 21702, ER-GenTech, Dipartimento di Biochimica e Biologia Molecolare, Molecular Biology Section,
UniVersita` di Ferrara, 44100 Ferrara, Italy
ReceiVed March 27, 2006
A new series of compounds in which the 2-amino-5-chlorophenyl ring of phenstatin analogue 7 was replaced
with a 2-amino-5-aryl thiophene was synthesized and evaluated for antiproliferative activity and for inhibition
of tubulin polymerization and colchicine binding to tubulin. 2-Amino-3-(3′,4′,5′-trimethoxybenzoyl)-5-phenyl
thiophene (9f) as well as the p-fluoro-, p-methyl-, and p-methoxyphenyl substituted analogues (9i, j, and l,
respectively) displayed high antiproliferative activities with IC50 values from 2.5 to 6.5 nM against the
L1210 and K562 cell lines. Compounds 9i and j were more active than combretastatin A-4 as inhibitors of
tubulin polymerization. Molecular docking simulations to the colchicine site of tubulin were performed to
determine the possible binding mode of 9i. The results obtained demonstrated that antiproliferative activity
correlated well with the inhibition of tubulin polymerization and the lengthening of the G2/M phase of the
cell cycle. Moreover, a good correlation was found between these inhibitory effects and the induction of
apoptosis in cells treated with the compounds.
Introduction
dynamics. CA-4 strongly inhibits the polymerization of tubulin
by binding to the colchicine site.5 CA-4 inhibits cell growth
even at very low (nanomolar) concentrations, exhibiting inhibi-
tory effects even on multidrug resistant (MDR) cancer cell lines.4
The disodium phosphate prodrug of CA-4 (CA-4P, 2) is water-
soluble, and there have been promising results with 2 as a tumor
vascular targeting agent in phase II clinical trials.6 Its structural
simplicity, along with its ability to selectively damage tumor
neovasculature, makes CA-4 of great interest from the medicinal
chemistry point of view. For these reasons, numerous CA-4
analogues have been synthesized and evaluated for their
structure-activity relationships (SAR).7 The replacement of the
olefinic bridge of CA-4 with a carbonyl group furnished a
benzophenone-type CA-4 analogue named phenstatin (3), which
has been found to be a potent cytotoxic agent and inhibitor of
tubulin polymerization, with activities differing little from those
of CA-4.8 Shifting the hydroxyl group from the C-3 to the C-2
position to furnish phenstatin isomer 4 dramatically decreased
antiproliferative activity.9 The replacement of the hydroxyl
moiety with an amino group at the C-2 position of the
benzophenone ring (compound 5) increased cytotoxic activity
by 100-fold compared with 4, indicating that the amino and
hydroxy groups are not bioequivalent at the C-2 position of these
phenstatin analogues. 2-Aminobenzophenone derivative 5 showed
significantly increased cytotoxicity against many human cancer
cell lines with activity similar to the activities of phenstatin 3
and CA-4.9 Changing the position of the methoxy group from
C-4 to C-5, as in compound 6, maintained antiproliferative
activity. The replacement of the C-5 methoxy group with a
chlorine atom, to give 7, had little effect on activity, whereas
its removal (8) reduced antiproliferative activity.10
Despite progress made in recent years in discovering new
compounds with antiangiogenic activity, this research field is
still considered one of the most promising for the development
of new ways to treat diseases characterized by abnormal
angiogenesis including tumors.1 Research oriented toward the
discovery of a new generation of agents useful in cancer
chemotherapy has identified tubulin as a possible molecular
target.2
The microtubule system of eukaryotic cells plays an important
role in regulating cell architecture, and it has an essential role
in cell division because microtubules are the key component of
the mitotic spindle. Microtubules are a dynamic cellular
compartment in both neoplastic and normal cells. This dyna-
micity is characterized by the continuous turnover of Râ-tubulin
heterodimers in the polymeric microtubules. The discovery of
natural and synthetic substances capable of interfering with the
polymerization or depolymerization of microtubules has attracted
much attention because microtubules are an attractive pharma-
cological target for anticancer drug discovery.2 More recently,
it was established that some tubulin binding agents also target
the vascular system of tumors, inducing morphological changes
in the endothelial cells of the tumor blood vessels so as to
occlude flow.3
Combretastatin A-4 (CA-4, 1), isolated from the bark of the
South African tree Combretum caffrum,4 is one of the well-
known natural tubulin binding molecules affecting microtubule
* To whom correspondence should be addressed. Phone: 39-(0)532-
291290. Fax: 39-(0)532-291296. E-mail: rmr@unife.it (R.R.). Phone: 39-
(0)532-291293. Fax: 39-(0)532-291296. E-mail: baraldi@unife.it (P.G.B.).
† Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
‡ SAIC-Frederick Inc., National Institutes of Health.
The benzene-thiophene pair represents a prominent example
of bioisosteres in drug research. Bioisosteres are groups of
molecules that are structurally similar and show the same type
§ Toxicology and Pharmacology Branch, National Institutes of Health.
| Dipartimento di Biochimica e Biologia Molecolare, Universita` di
Ferrara.
10.1021/jm060355e CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/27/2006

2-Amino-3-(3′,4′,5′-trimethoxybenzoyl)-5-aryl Thiophenes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3907
of biological activity. In the case of the 2-aminobenzene moiety,
the C-3/C-4 ethylene group (-CHdCH-) can be replaced by a
divalent sulfur (-S-) atom, producing a 2-aminothiophene. Then
the C-4 and C-5 positions of the thiophene moiety (see general
structure 9) can be considered equivalent to the C-6 and C-5
positions, respectively, of the 2-aminobenzene moiety, numbered
as in compounds 5-8. The classical bioisosteric equivalence
between benzene and thiophene prompted us to synthesize a
series of 2-amino-3-(3′,4′,5′-trimethoxybenzoyl)-thiophene de-
rivatives with general formula 9 in which a 2-aminothiophene
system replaced the 2-aminobenzene moiety of compounds 5-8.
The potent activity of C-5 methoxy and chloro derivatives 6
and 7 led us to synthesize a series of thiophene derivatives with
various substituents at C-5 equivalent to the phenyl C-5 position
of 5-8. Our goal was to evaluate the steric and electronic effects
of the substituents on antiproliferative activity. Besides hydrogen
(compound 9a), the substituents examined included halogens
(bromine in 9b and iodine in 9c), alkyl groups (ethyl and
n-pentyl, 9d and e, respectively), and phenyl (9f) and substituted
phenyl groups (9g-o). The latter compounds were the most
active members of the series, and thus, we were able to analyze
electron-withdrawing (F, Cl, and CF3) and electron-donating
(Me and MeO) substituents on the C-5 phenyl ring as well as
positional effects with three methoxyl group isomers (9l-n).
In addition, we synthesized and examined thiophene derivatives
with C-4 substituents (methyl and phenyl, compounds 9p and
q, respectively) and three (9r-t) 4,5-disubstituted thienyl
analogues.
Chemistry. 2-Amino-3-(3′,4′,5′-trimethoxybenzoyl)thiophene
derivatives with general structure 9 were synthesized as shown
in the reaction sequence reported in Scheme 1. 2-Amino-3-aroyl
thiophenes 9a, d-f, p, r, and u and v were obtained by a one-
step procedure (Gewald reaction12) applied to â-ketonitriles
10a-c13 and the appropriate aldehyde or ketone with sulfur and
triethylamine (TEA) as base in refluxing ethanol. Compound
9q was synthesized by a two-step procedure consisting of a
Knoevenagel reaction of 10a and acetophenone in benzene in
the presence of â-alanine and acetic acid, followed after isolation
and purification of the E- and Z-olefin isomers, by a cyclization
in ethanol with sulfur in the presence of TEA.14 The 5-unsub-
stituted thiophene derivatives 9a, p, and q were transformed
almost quantitatively into the corresponding N-phthalimido
derivatives 11a-c using phthalic anhydride in refluxing acetic
acid.15 The subsequent regioselective bromination of 11a-c in
a mixture of acetic acid and sodium acetate, using a stoichio-
metric amount of bromine,16 furnished intermediate 5-bro-
mothiophene derivatives 12a-c in good yields. The formation
of the 4-bromothiophene isomer of compound 11a was not
observed. The preparation of the thiophene 5-iodo substitution
12d was carried out by stirring 11a in acetic acid with
N-iodosuccinimide (NIS) at 50 °C.17
The eleven 5-aryl thiophene compounds 13g-o and s and t
were synthesized from key intermediates 12a-c by a standard
Suzuki cross-coupling reaction with appropriate aryl boronic
acids under heterogeneous conditions (Pd(PPh3)4, K2CO3) in
refluxing toluene.18 The removal of the N-protected phthaloyl
group was accomplished by the use of hydrazine in refluxing
ethanol, to afford final compounds 9b and c, g-o, and s and
t.19
The removal of either the C′-3 or the C′-4 methoxy group of
CA-4 causes a substantial loss of cytotoxicity,11 and we
demonstrated, through the syntheses of compounds 9u and v,
that a similar loss of antiproliferative activity occurs in our
thiophene series relative to the 2-amino-5-phenylthiophene
derivative 9f.
Results and Discussion
Effects on in Vitro Proliferation of the K562 and L1210
Tumor Cell Lines. Table 1 summarizes the effects of thiophene
derivatives 9a-v on the growth of murine L121020 and human
K56221a leukemia cells with CA-4 (1) as the reference com-
pound. L1210 and K562 cell lines are among the most used
tumor cell lines for the screening of potential antitumor
compounds.21b,c Seven compounds had activities in both cell
lines comparable with the activities of CA-4, but most of the
other compounds were minimally cytotoxic.
The 4,5-unsubstituted thiophene derivative 9a displayed only
modest antiproliferative activity, which was little affected with
alkyl (ethyl, 9d or n-pentyl, 9e) or halogen (Br, 9b or I, 9c)
substituents at C-5. In contrast, 9 out of 10 compounds with
aryl substituents (9f-o, the exception being 9o) at C-5 had
significantly improved cytotoxicity relative to that of 9a. In fact,
6 of the 7 most cytotoxic compounds were in this group. The
contribution of the phenyl group to activity, as in 9f, was
position-dependent. Placing the phenyl at C-4 (9q) resulted in
an inactive compound. The substitution on the phenyl ring had
variable effects. A para fluoro group (9i) may have slightly
improved antiproliferative activity, and all other para substituents
prepared (Cl (9g), CH3 (9j), OCH3 (9l)), except p-CF3 (9k),
had minimal effects relative to those of 9f. Only a slight loss
of activity was observed when the methoxy substituent was
moved from the para position to either the ortho (9n) or the
meta (9m) position. However, either two (9h) or three (9o)
substituents on the C-5 phenyl group led to a significant loss
of antiproliferative activity, suggesting that steric factors account
for the loss of activity observed with these two compounds.
These data show no clear steric or electronic effects, except
that multiple substituents on the phenyl ring or the strong

3908 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Romagnoli et al.
Scheme 1a
a Reagents: a: ketone or aldehyde, S8, TEA, EtOH, 70 °C for 2 h; for 9q: acetophenone, 10a, AcOH, â-alanine, benzene, rx, 16 h; b: phthalic anhydride,
AcOH; c: Br2, AcOH-AcONa for 11 a-c, r.t.; NIS, AcOH, 50 °C; d: ArB(OH)2, Pd(PPh3)4, Na2CO3, PhMe, rx, 18 h; e: NH2NH2, EtOH.
electron-withdrawing group CF3 were present in the least active
C-5 aryl substituents.
of 9f. Its substitution with the 3′,4′-dimethoxybenzoyl and,
especially, 4′-methoxybenzoyl moieties (compounds 9u and v,
respectively), led to the loss of antiproliferative activity.
Considering the substitution at the thiophene C-4 position,
substituent size seemed to be a factor related to antiproliferative
activity. The weak micromolar activity of 2-amino-3-(3′,4′,5′-
trimethoxybenzoyl) thiophene (9a) was enhanced up to 12-fold
(in the L1210 cell line) by the 4-methyl substitution (derivative
9p) and eliminated by a 4-phenyl substituent (9q).
Starting from 9f, with the C-5 phenyl substituent, the insertion
of a second phenyl ring at C-4 yielded an inactive compound
(9t). The steric hindrance of the C-4 phenyl group should result
in conformational changes in the relative positions of the 3′,4′,5′-
trimethoxybenzoyl and phenyl moieties at the C-3 and C-5
positions, respectively, and this could cause the inactivity of
9t. In support of this idea, a methyl group at C-4 in the 9f
structure (i.e., compound 9s) led to a small loss of cytotoxic
activity, whereas the replacement of the C-5 phenyl ring in 9s
with a methyl group (compound 9r) led to a significant loss of
activity.
Effects on Tubulin Polymerization and on Colchicine
Binding to Tubulin. To investigate whether the antiproliferative
activities of these compounds were related to the interaction
with the microtubule system, seven of the most active com-
pounds (9f and g, 9i and j, 9l and m, and 9s) were evaluated
for the inhibition of the polymerization of purified tubulin and
the binding of [3H]colchicine to tubulin (Table 2).22a,b For
comparison, the data of the potent antimitotic compounds CA-4
and podophyllotoxin are also presented. The results are con-
sistent with the conclusion that tubulin is the intracellular target
of these seven compounds because they were all potent
inhibitors of both tubulin-dependent reactions with activities
quantitatively similar to those of CA-4 and podophyllotoxin.
Three compounds, 9g and 9i and j, were better inhibitors of
assembly than either of the standard drugs, all seven compounds
were better inhibitors of colchicine binding than podophyllo-
toxin, and the most cytotoxic of the thiophene derivatives, 9i,
was as potent as CA-4 as an inhibitor of colchicine binding, a
property rarely observed in colchicine site drugs.
Synthesis of derivatives 9u and v, both significantly less
active than 9f, demonstrated that the presence of the 3′,4′,5′-
trimethoxybenzoyl moiety was essential for the optimal activity

2-Amino-3-(3′,4′,5′-trimethoxybenzoyl)-5-aryl Thiophenes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3909
Table 1. In Vitro Activity of Compounds 9a-v and CA-4 (1) against
the Proliferation of Murine L1210 and Human K562 Leukemia Cell
Lines
IC50 (nM)a
compd
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
K562
L1210
1267 ( 289
1677 ( 380
342 ( 43
9000 ( 707
1490 ( 210
3.75 ( 1.56
14.7 ( 1.52
362 ( 34
3150 ( 1900
4250 ( 1060
1150 ( 490
>10,000
1325 ( 106
2.52 ( 0.70
6.96 ( 0.36
337 ( 18
2.15 ( 1.26
3.52 ( 0.36
73.7 ( 8.83
6.52 ( 0.35
20.8 ( 1.26
9.00 ( 2.82
1650 ( 630
262 ( 3.52
>10000
2.82 ( 1.82
4.72 ( 1.34
70.3 ( 14.1
4.47 ( 1.70
10.1 ( 3.36
7.75 ( 2.12
1100 ( 100
219 ( 26
>10000
790 ( 230
6.42 ( 3.07
8500 ( 356
85.5 ( 9.12
4670 ( 290
3.6 ( 1.4
3900 ( 140
18.2 ( 2.20
6950 ( 235
537 ( 18.8
>10000
9s
9t
9u
9v
CA-4 (1)
8.3 ( 2.1
a IC50 is the 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.
Table 2. Inhibition of Tubulin Polymerization and Colchicine Binding
by Compounds 9f and g, i and j, l and m, and s
tubulin
assemblya
IC50 ( SD
(µM)
colchicine
bindingb
% ( SD
compd
Figure 1. Effects of compounds 9f, i, and j on apoptosis in K562
cells. The cells were cultured for 4 days without any compounds (A)
or with compounds 9f (B), 9i (C), and 9j (D) leading to 50% inhibition
of cell growth. In panels E and F, enlargements of the areas represented
by the boxes in panels A and B, respectively, are shown. Representative
apoptotic cells are indicated by arrows in panel F.
9f
9g
9i
9j
9l
1.9 ( 0.04
0.98 ( 0.10
1.1 ( 0.00
1.1 ( 0.04
1.7 ( 0.00
1.7 ( 0.00
2.2 ( 0.2
60 ( 5
68 ( 2
82 ( 3
71 ( 1
64 ( 1
58 ( 4
37 ( 2
34 ( 10
82 ( 0.3
9m
9s
of sub-G1 cells. In the first experiment (Figure 2, panels A-C)
in which the cells were treated with 3 and 10 nM 9f, there was
a clear increase of the proportion of sub-G1 cells, indicative of
extensive DNA breakage. Figure 2 (panels D-G) demonstrates
that compounds 9i and j as well as f induce a sharp increase in
the proportion of sub-G1 cells. Thus, by two independent
methods, we show that the most active thiophene derivatives
cause apoptosis and, as expected from their antitubulin proper-
ties, are cytotoxic rather than cytostatic compounds.
Analysis of Cell Cycle. Because molecules exhibiting activity
on tubulin binding should cause the alteration of cell cycle
parameters leading to a preferential G2/M blockade,5-7 the
effects of the most active compound of the series (9i) on cell
cycle distribution were analyzed in K562 cells cultured for 24
h in the presence of increasing amounts of the compound. The
results of a representative experiment (Figure 3) show a dose-
dependent increase in the proportion of G2-M cells occurring
with a simultaneous decrease in S and G1 cells. As expected
from the results shown in Figure 2, sub-G1 cells are found in
K562 cells treated with the highest amount of 9i. Similar effects
on the cell cycle were obtained using the other active compounds
(data not shown), suggesting that this class of molecules act,
as expected from the data shown in Table 2, selectively on the
G2/M phase of the cell cycle.
Podophyllotoxin
CA-4 (1)
1.7 ( 0.1
1.7 ( 0.2
a Inhibition of tubulin polymerization. Tubulin was at 10 µM. b Inhibition
of [3H]colchicine binding. Tubulin, colchicine, and the test compound were
at 1, 5, and 1 µM concentration, respectively.
We conclude that the antiproliferative activity of these
compounds derives from an interaction with the colchicine site
of tubulin and an interference with microtubule assembly. This
indicates that the cellular actions of these agents would involve
mitotic arrest due to the interference with the functions of the
mitotic spindle and apoptotic cell death.23-26
Effects on Apoptosis. We determined whether compounds
exhibiting high antitubulin activity induce apoptosis of K562
cells. The cells were treated with the most potent antiprolifera-
tive compounds and analyzed by light microscopy to determine
the percentage of cells displaying an apoptotic phenotype. Figure
1 shows a representative image of K562 cells treated with 9f,
i, and j. These three compounds displayed the highest levels of
apoptosis among the compounds examined. The average values
of the percentage of apoptotic cells induced by these compounds
were 87.5 ( 4.3% (9f), 20.5 ( 5.2% (9j), and 64.5 ( 6.2 (9i)
in three independent experiments.
In agreement with the microscopic evaluation, the flow
cytometric evalution of cells following treatment with com-
pounds 9f, i, and j showed the appearance of substantial numbers
Molecular Modeling Studies. Molecular docking simulations
to the colchicine site of tubulin were performed to determine a

3910 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Romagnoli et al.
Figure 2. Effects of compounds 9f (B, C, E), 9i (F), and 9j (G) on DNA content/cell following the treatment of K562 cells for 4 days. The cells
were cultured without any compounds (A, D) or with compounds 9f, i, or j leading to 50% (B) and 75% (C, E-G) inhibition of cell growth. Cell
cycle distribution was analyzed by the standard propidium iodide procedure as described in the Experimental Section. Sub-G1, G1, S, and G2 cells
are indicated.
Figure 3. Effects of compound 9i on the cell cycle distribution of K562 cells. The cells were cultured for 24 h without the compound (A) or
withcompound 9i leading to 25% (B), 50% (C), and 75% (D) inhibition of cell growth. Cell cycle distribution was analyzed by the standard
propidium iodide procedure as described in the Experimental Section. Sub-G1, G1, S and G2 cells are indicated. Quantitative analysis of the data
shown in panels A-D (E).
possible binding mode of 9i. The recently reported crystal-
lographic 3D structure of tubulin was retrieved from the Protein
Data Bank.27 All docking runs were performed to the colchicine
site of the macromolecule, applying the Lamarckian genetic
algorithm (LGA) of AutoDock 3.0.28
colchicine (DAMA-colchicine) was redocked into the binding
site of the protein using the above algorithm. The pose of the
original X-ray crystallographic reference structure was success-
fully reproduced (rmsd: 0.79 Å, ∆Gbind ) -12.07 kcal/mol).
Concerning compound 9i, the calculated free energies of
binding were used as the parameter for the selection of the
cluster of docking poses to be evaluated. The docked ligand
To set a consistent flexible docking protocol for compound
9i, the cocrystallized ligand N-deacetyl-N-(2-mercaptoacetyl)-

2-Amino-3-(3′,4′,5′-trimethoxybenzoyl)-5-aryl Thiophenes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3911
molecular skeleton of 2-amino-3-(3′,4′,5′-trimethoxybenzoyl)-
thiophene. The compounds were evaluated for their antiprolif-
erative activities against both L1210 and K562 cells. Seven of
the 22 new compounds displayed potent activity, differing little
from that of CA-4. SAR studies showed that the most active
analogues had a phenyl or a substituted phenyl group in the
5-position of the thiophene ring along with a hydrogen or a
small substituent (methyl, compound 9s) in the 4-position.
Indeed, the absence of a 5-aryl moiety (9a), including its
replacement by a halogen (9b and c) or an alkyl substituent
(9d and e), led to sharply reduced activity. The investigations
reported here were aimed at the elucidation of the mechanism
of action by which these compounds exert their antiproliferative
activity. As the molecular target of the compounds, we identified
tubulin, the major constituent of the cellular microtubule system.
Six of the seven most cytotoxic members of the series were all
evaluated for effects on tubulin assembly and inhibition of
colchicine binding. The IC50 values in a relatively narrow range
(1-2.2 µM) were obtained for 9f and g, i and j, l and m, and
9s as inhibitors of tubulin assembly. The three most potent
inhibitors of tubulin assembly (9g and i and j) also exhibited
high levels of induction of apoptosis and DNA breakage.
Compound 9i, with the highest antiproliferative activity, was
the strongest inhibitor both of tubulin assembly and [3H]-
colchicine binding to tubulin. The results of docking studies
are consistent with the observed biological data for compound
9i. The binding of this derivative to tubulin was stabilized by
hydrogen-bonding interactions with Leu-252, Asn-258, and Gln-
11 of â-tubulin. Cell-cycle distribution analysis shows that 9i,
as expected from its inhibition of tubulin assembly, acts on the
G2/M phase of the cell cycle. These derivatives constitute an
interesting new class of antitubulin agents with the potential to
be clinically developed for cancer treatment. Among their
attractive properties are the ease of synthesis and chemical
stability.
Figure 4. DAMA-colchicine and 9i docked into the binding site of
tubulin. (The Connoly surface is shown in gray.) The carbon atoms of
DAMA-colchicine and 9i are green and violet, respectively. The
Connoly surface of the region along the z-axis is, on purpose, not
highlighted.
Experimental Section
Chemistry. Materials and Methods. 1-Phenylethanone, 2,5-
dihydroxy-1,4-dithiane, 2,5-dihydroxy-2,5-dimethoxy-1,4-dithiane,
butanal, heptanal, phenylacetaldehyde, and 2-butanone are com-
Figure 5. Compound 9i (carbon atom are violet) docked into the
tubulin binding pocket. Both side chains of important active site amino
acids and hydrogen bonds are shown: Leu-252 (angle N-H...O )
132.74°, distance ) 2.675 Å), Asn-258 (angle O...H-N ) 162.16°,
distance ) 2.716 Å), and Gln-11 (angle CdO...F ) 136.08°, distance
) 2.545 Å). Amino acid residue numbers are as in the crystal structure.27
1
mercially available and used as received. H NMR spectra were
recorded on a Bruker AC 200 spectrometer. Chemical shifts (δ)
are given in ppm, upfield from tetramethylsilane as the 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 uncorrected. All products reported
showed 1H NMR spectra in agreement with the assigned structures.
Elemental analyses were conducted by the Microanalytical Labora-
tory of the Chemistry Department of the University of Ferrara. All
reactions were carried out under an inert atmosphere of dry nitrogen,
unless otherwise described. Standard syringe techniques were
applied for transferring dry solvents. Reaction courses and product
mixtures were routinely monitored by TLC on silica gel (precoated
F254 Merck plates) and visualized with aqueous KMnO4. Flash
chromatography was performed using 230-400 mesh silica gel and
the indicated solvent system. Organic solutions were dried over
anhydrous Na2SO4. Calcium hydride and calcium chloride were
used in the distillation of dioxane and DMF, respectively, and the
distilled solvents were stored over molecular sieves (3 Å). In high-
pressure hydrogenation experiments, a Parr shaker on a high-
pressure autoclave was used.
showed a similar orientation to that of DAMA-colchicine in
the tubulin binding pocket. Subsequently, the selection of the
putative bioactive conformation was performed mainly on a
geometrical basis, namely, the conformation with the best fit
to the common trimethoxybenzoyl moiety in terms of rmsd. The
selected pose of 9i had an estimated free energy of binding of
-10.11 kcal/mol, and the trimethoxybenzoyl ring of the ligand
superimposed well on the same moiety of DAMA-colchicine
(Figure 4). This finding is in agreement with data reported in
the literature concerning the possible binding mode of other
potent colchicine site inhibitors of tubulin polymerization.29,30
Moreover, the binding of compound 9i to tubulin was
stabilized by hydrogen bonding interactions with Leu-252, Asn-
258, and Gln-11 of â-tubulin (Figure 5). The results of our
docking studies are consistent with the high inhibitory activity
observed for compound 9i. The residue numbering system used
here is from the crystal structure.27
Synthesis of (2-Amino-4-phenylthiophen-3-yl)-(3,4,5-trimethoxy-
phenyl)-methanone (9q). A mixture of 3-oxo-3-(3,4,5-trimethoxy-
phenyl)-propionitrile (10a) (588 mg, 2.5 mmol), 1-phenylethanone
(2.5 mmol), â-alanine (0.5 mmol), acetic acid (0.6 mL), and benzene
(15 mL) was refluxed under Dean-Stark conditions for 16 h. The
mixture was cooled, washed with water (5 mL) and brine (5 mL),
Conclusions
In conclusion, we have discovered a new class of simple
synthetic inhibitors of tubulin polymerization based on the

3912 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Romagnoli et al.
dried, evaporated, and purified by column chromatography (EtOAc-
petroleum ether 3:7) to furnish the E/Z mixture of Knoevenagel’s
adduct as a yellow oil (48% yield). The oil was dissolved in EtOH
(5 mL), and sulfur (64 mg, 2 mmol) and TEA (250 µL, 2 mmol)
were added. The mixture was stirred for 2 h at 50 °C. The mixture
was cooled to r.t. and evaporated, and the residue was dissolved in
dichloromethane (DCM, 10 mL), washed with water (2 mL) and
brine (2 mL), dried, evaporated, purified by column chromatography
(EtOAc-petroleum ether 3:7), and crystallized from petroleum
ether. Yellow solid (55% yield); mp 154-156 °C. 1H NMR
(CDCl3): δ 3.69 (s, 3H), 3.71 (s, 6H), 6.22 (bs, 2H), 6.59 (s, 2H),
7.02 (m, 6H). Anal. (C20H19NO4S): C, H, N.
General Procedure A for the Synthesis of Compounds 9a,
d-f, p-r, u and v. To a suspension of aroylacetonitrile derivative
10a, b, or c (5 mmol), TEA (0.44 mL, 5 mmol), and sulfur (164
mg, 5 mmol) in EtOH (10 mL) was added the appropriate ketone
or aldehyde (5 mmol). After stirring for 2 h at 70 °C, the solvent
was evaporated and the residue diluted with DCM (15 mL). After
washing with water (2 × 5 mL) and brine (5 mL), the organic
layer was dried and evaporated. The crude product was purified
by column chromatography and crystallized from petroleum ether.
(2-Aminothiophen-3-yl)-(3,4,5-trimethoxyphenyl)-methan-
one (9a). 2,5-Dihydroxy-1,4-dithiane (thioacetaldehyde dimer),
3-oxo-3-(3,4,5-trimethoxyphenyl)-propionitrile (10a), TEA, and
sulfur were reacted according to general procedure A. Purification
by silica gel column chromatography (petroleum ether-EtOAc 3:7)
afforded 9a as a yellow powder (74% yield); mp 140-142 °C. 1H
NMR (CDCl3): δ 3.89 (s, 3H), 3.91 (s, 6H), 6.16 (d, J ) 6.2 Hz,
1H), 6.28 (bs, 2H), 6.94 (s, 2H), 7.00 (d, J ) 6.2 Hz, 1H). Anal.
(C14H15NO4S): C, H, N.
(2-Amino-5-ethylthiophen-3-yl)-(3,4,5-trimethoxyphenyl)-meth-
anone (9d). Butanal, 3-oxo-3-(3,4,5-trimethoxyphenyl)-propionitrile
(10a), TEA, and sulfur were reacted according to general procedure
A. Purification by silica gel column chromatography (petroleum
ether-EtOAc 2:8) afforded 9d as a yellow oil (65% yield). 1H NMR
(CDCl3): δ 1.21 (t, J ) 7.6 Hz, 3H), 2.62 (q, J ) 7.6 Hz, 2H),
3.89 (s, 3H), 3.90 (s, 6H), 6.33 (bs, 2H), 6.59 (s, 2H), 6.94 (s, 1H).
Anal. (C16H19NO4S): C, H, N.
(2-Amino-5-phenylthiophen-3-yl)-(3,4-dimethoxyphenyl)-meth-
anone (9u). Phenylacetaldehyde (50% solution in water), 3-oxo-
3-(3,4-dimethoxyphenyl)-propionitrile (10b), TEA, and sulfur were
reacted according to general procedure A. Purification by silica
gel column chromatography (petroleum ether-EtOAc 3:7) afforded
1
afforded 9u as a yellow solid (66% yield); mp 112-114 °C. H
NMR (CDCl3): δ 3.93 (s, 3H), 3.95 (s, 3H), 6.28 (bs, 2H), 6.91
(s, 1H), 6.95 (d, J ) 7.6 Hz, 1H), 7.20 (s, 1H), 7.33 (m, 6H). Anal.
(C19H17NO3S): C, H, N.
(2-Amino-5-phenylthiophen-3-yl)-(4-methoxyphenyl)-metha-
none (9v). Phenylacetaldehyde, 3-oxo-3-(4-methoxyphenyl)-pro-
pionitrile (10c), TEA, and sulfur were reacted according to general
procedure A. Purification by silica gel column chromatography
(petroleum ether-EtOAc 3:7) afforded 9v as an orange gum (56%
1
yield). H NMR (CDCl3): δ 3.88 (s, 3H), 6.94 (bs, 2H), 6.99 (s,
1H), 7.32 (m, 7H), 7.73 (d, J ) 8.8 Hz, 2H). Anal. (C18H15NO2S):
C, H, N.
General Procedure B for the Preparation of Compounds
11a-c. To a suspension of compound 9a, p, or q (6 mmol) in
acetic acid (20 mL) was added phthalic anhydride (7.2 mmol, 1.07
g). After stirring for 18 h at reflux, the solvent was evaporated and
the residue dissolved in EtOAc (30 mL). The organic solution was
washed with a saturated solution of NaHCO3 (10 mL), water (10
mL), and brine (10 mL), dried, and concentrated. The crude product
was purified by crystallization from petroleum ether.
2-[3-(3,4,5-Trimethoxybenzoyl)-thiophen-2-yl]-isoindole-1,3-
dione (11a). Following general procedure B, starting from com-
pound 9a, derivative 11a was obtained as a yellow solid (75%
1
yield); mp 153-155 °C. H NMR (CDCl3): δ 3.72 (s, 3H), 3.83
(s, 6H), 6.98 (s, 2H), 7.41 (m, 2H), 7.77 (m, 2H), 7.84 (m, 2H).
2-[4-Methyl-3-(3,4,5-trimethoxybenzoyl)-thiophen-2-yl]-isoin-
dole-1,3-dione (11b). Following general procedure B, starting from
compound 9p, derivative 11b was obtained as a yellow solid (60%
1
yield); mp 108-110 °C. H NMR (CDCl3): δ 2.34 (s, 3H), 3.61
(s, 3H), 3.79 (s, 6H), 6.91 (s, 2H), 7.08 (s, 1H), 7.70 (m, 4H).
2-[4-Phenyl-3-(3,4,5-trimethoxybenzoyl)-thiophen-2-yl]-isoin-
dole-1,3-dione (11c). Following general procedure B, starting from
compound 9q, derivative 11c was obtained as a yellow solid (68%
1
yield); mp 152-154 °C. H NMR (CDCl3): δ 3.62 (s, 3H), 3.82
(2-Amino-5-pentylthiophen-3-yl)-(3,4,5-trimethoxyphenyl)-
methanone (9e). Heptanal, 3-oxo-3-(3,4,5-trimethoxyphenyl)-pro-
pionitrile (10a), TEA, and sulfur were reacted according to general
procedure A. Purification by silica gel column chromatography
(petroleum ether-EtOAc 2:8) afforded 9e as a yellow oil (61%
(s, 6H), 6.92 (s, 2H), 7.12 (m, 6H), 7.62 (m, 2H), 7.72 (m, 2H).
General Procedure C for the Preparation of Compounds
12a-c. To an acetic acid solution (12 mL) of derivatives 11a, b,
or c (5 mmol) containing sodium acetate (5.5 mmol), bromine (5
mmol) was added dropwise with stirring at r.t over 5 min. After 1
h, the acetic acid was removed in vacuo, and the crude residue
diluted with EtOAc (25 mL), washed with an aqueous 10% sodium
thiosulfate solution (10 mL), a saturated solution of NaHCO3 (10
mL), water (5 mL), and brine (5 mL), and it was dried and
concentrated. The resulting residue was recrystallized from petro-
leum ether.
1
yield). H NMR (CDCl3): δ 1.31 (m, 9H), 2.63 (t, J ) 7.6 Hz,
2H), 3.87 (s, 3H), 3.88 (s, 6H), 6.34 (bs, 2H), 6.61 (s, 2H), 6.90 (s,
1H). Anal. (C19H25NO4S): C, H, N.
(2-Amino-5-phenylthiophen-3-yl)-(3,4,5-trimethoxyphenyl)-
methanone (9f). Phenylacetaldehyde (50% solution in water),
3-oxo-3-(3,4,5-trimethoxyphenyl)-propionitrile (10a), triethylamine,
and sulfur were reacted according to general procedure A. Purifica-
tion by silica gel column chromatography (toluene-ethyl ether 7:3)
afforded 9f as a yellow solid (78% yield); mp 63-64 °C. 1H NMR
(CDCl3): δ 3.89 (s, 3H), 3.92 (s, 6H), 6.26 (bs, 2H), 6.97 (s, 2H),
7.18 (s, 1H), 7.36 (m, 5H). Anal. (C20H19NO4S): C, H, N.
2-[5-Bromo-3-(3,4,5-trimethoxybenzoyl)-thiophen-2-yl]-isoin-
dole-1,3-dione (12a). Yellow solid (82% yield); mp 175-177 °C.
1H NMR (CDCl3): δ 3.69 (s, 3H), 3.83 (s, 6H), 6.94 (s, 2H), 7.37
(s, 1H), 7.73 (m, 2H), 7.84 (m, 2H).
2-[5-Bromo-4-methyl-3-(3,4,5-trimethoxybenzoyl)-thiophen-
2-yl]-isoindole-1,3-dione (12b). Yellow solid (86% yield); mp
183-184 °C. 1H NMR (CDCl3): δ 2.33 (s, 3H), 3.62 (s, 3H), 3.80
(s, 6H), 6.96 (bs, 2H), 7.67 (m, 2H); 7.74 (m, 2H).
(2-Amino-4-methylthiophen-3-yl)-(3,4,5-trimethoxyphenyl)-
methanone (9p). 2,5-Dimethyl-2,5-dihydroxy-1,4-dithiane, 3-oxo-
3-(3,4,5-trimethoxyphenyl)-propionitrile (10a), TEA, and sulfur
were reacted according to general procedure A. Purification by silica
gel column chromatography (petroleum ether-EtOAc 2:8) afforded
2-[5-Bromo-4-phenyl-3-(3,4,5-trimethoxybenzoyl)-thiophen-
2-yl]-isoindole-1,3-dione (12c). Yellow solid (71% yield); mp 163-
1
9p as a yellow powder (50% yield); mp 148-150 °C. H NMR
1
(CDCl3): δ 1.82 (s, 3H), 3.89 (m, 9H), 5.86 (s, 1H), 6.43 (bs, 2H),
6.77 (s, 2H). Anal. (C15H17NO4S): C, H, N
165 °C. H NMR (CDCl3): δ 3.61 (s, 3H), 3.78 (s, 6H), 6.90 (s,
2H), 7.08 (m, 5H), 7.60 (m, 2H), 7.68 (m, 2H).
(2-Amino-4,5-dimethylthiophen-3-yl)-(3,4,5-trimethoxyphenyl)-
methanone (9r). 2-Butanone, 3-oxo-3-(3,4,5-trimethoxyphenyl)-
propionitrile (10a), TEA, and sulfur were reacted according to
general procedure A. Purification by silica gel column chromatog-
raphy (petroleum ether-EtOAc 3:7) afforded 9r as a yellow powder
2-[5-Iodo-3-(3,4,5-trimethoxybenzoyl)-thiophen-2-yl]-isoindole-
1,3-dione (12d). A mixture of derivative 11a (1 mmol) and NIS
(4.4 mmol, 1.1 equiv) in acetic acid was heated at 50 °C for 6 h.
Acetic acid was removed under reduced pressure, the crude residue
was diluted with EtOAc (25 mL), washed with a saturated solution
of NaHCO3 (10 mL), water (5 mL), and brine (5 mL), and dried
and concentrated. The resulting residue was recrystallized from
petroleum ether. Compound 12d was obtained as a brown solid
1
(68% yield); mp 78-80 °C. H NMR (CDCl3): δ 1.65 (s, 3H),
2.14 (s, 3H), 3.86 (s, 6H), 3.89 (s, 3H), 6.28 (bs, 2H), 6.78 (s, 2H).
Anal. (C16H19NO4S): C, H, N.

2-Amino-3-(3′,4′,5′-trimethoxybenzoyl)-5-aryl Thiophenes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3913
(38% yield); mp 179-181 °C. 1H NMR (CDCl3): δ 3.38 (s, 3H),
3.68 (s, 6H), 6.81 (s, 2H), 7.65 (s, 1H), 7.84 (m, 4H).
Yellow solid (60% yield); mp 198-200 °C. 1H NMR (CDCl3): δ
3.73 (s, 3H), 3.86 (s, 9H), 6.98 (m, 2H), 7.00 (s, 2H), 7.15 (m,
1H), 7.37 (m, 1H), 7.56 (s, 1H), 7.78 (m, 2H), 7.85 (m, 2H).
2-[3-(3,4,5-Trimethoxybenzoyl)-5-(3,4,5-trimethoxyphenyl)-
thiophen-2-yl]-isoindole-1,3-dione (13o). The title compound was
prepared by general procedure D from 12a and m,m′,p-trimethoxy-
phenylboronic acid. EtOAc-petroleum ether (4:6) was used for
flash chromatography. Yellow solid (60% yield); mp 181-182 °C.
1H NMR (CDCl3): δ 3.66 (s, 3H), 3.82 (s, 6H), 3.89 (s, 3H), 3.92
(s, 6H), 6.82 (s, 1H), 6.98 (s, 2H), 7.50 (s, 2H), 7.74 (m, 2H), 7.85
(m, 2H).
General Procedure D (Suzuki Coupling) for the Synthesis of
Compounds 13g-o, s, and t. A mixture of thiophene derivatives
12a, b, or c (0.5 mmol), potassium carbonate (104 mg, 0.75 mmol,
1.5 equiv), the appropriate aryl boronic acid (1 mmol, 2 equiv),
and tetrakis(triphenylphosphine)palladium (13.5 mg, 0.012 mmol)
in dry toluene (10 mL) was stirred at 100 °C under nitrogen for 18
h, cooled to ambient temperature, and evaporated in vacuo. The
residue was dissolved with EtOAc (30 mL), and the resultant
solution was washed sequentially with 5% NaHCO3 (10 mL), water
(10 mL), and brine (10 mL). The organic layer was dried, filtered,
and evaporated, and the residue was purified by flash chromatog-
raphy on silica gel.
2-[5-(4-Chlorophenyl)-3-(3,4,5-trimethoxybenzoyl)-thiophen-
2-yl]-isoindole-1,3-dione (13g). The title compound was prepared
by general procedure D from 12a and p-chlorophenylboronic acid.
EtOAc-petroleum ether (2:8) was used for flash chromatography.
Yellow solid (90% yield); mp 216-218 °C. 1H NMR (CDCl3): δ
3.62 (s, 3H), 3. 76 (s, 6H), 6.92 (s, 2H), 7.19 (s, 1H), 7.35 (d, J )
8.8 Hz, 2H), 7. 50 (d, J ) 8.8 Hz, 2H), 7. 51 (s, 1H), 7.74 (m,
2H), 7.77 (m, 2H).
2-[5-(3,4-Dichlorophenyl)-3-(3,4,5-trimethoxybenzoyl)-thiophen-
2-yl]-isoindole-1,3-dione (13h). The title compound was prepared
by general procedure D from 12a and m,p-dichlorophenylboronic
acid. EtOAc-petroleum ether (2:8) was used for flash chromatog-
raphy. Yellow solid (70% yield); mp 225-226 °C. 1H NMR
(CDCl3): δ 3.72 (s, 3H), 3.84 (s, 6H), 6.97 (s, 2H), 7.45 (m, 3H),
7. 51 (s, 1H), 7.74 (m, 2H), 7.79 (m, 2H).
2-[5-(4-Fluorophenyl)-3-(3,4,5-trimethoxybenzoyl)-thiophen-
2-yl]-isoindole-1,3-dione (13i). The title compound was prepared
by general procedure D from 12a and p-fluorophenylboronic acid.
EtOAc-petroleum ether (2:8) was used for flash chromatography.
Yellow solid (85% yield); mp 198-200 °C. 1H NMR (CDCl3): δ
3.72 (s, 3H), 3.83 (s, 6H), 7.00 (s, 2H), 7.65 (s, 1H), 7.70 (m, 4H),
7.84 (m, 2H), 7.86 (m, 2H).
2-[4-Methyl-5-phenyl-3-(3,4,5-trimethoxybenzoyl)-thiophen-
2-yl]-isoindole-1,3-dione (13s). The title compound was prepared
by general procedure D from 12b and phenylboronic acid. EtOAc-
petroleum ether (2:8) was used for flash chromatography. Yellow
1
cream solid (62% yield); mp 143-144 °C. H NMR (CDCl3): δ
2.33 (s, 3H), 3.62 (s, 3H), 3.82 (s, 6H), 6.98 (s, 2H), 7.48 (m, 5H),
7.75 (m, 4H).
2-[4,5-Diphenyl-3-(3,4,5-trimethoxybenzoyl)-thiophen-2-yl]-
isoindole-1,3-dione (13t). The title compound was prepared by
general procedure D from 12c and phenylboronic acid. EtOAc-
petroleum ether (2:8) was used for flash chromatography. Yellow
solid (71% yield); mp 167-169 °C. 1H NMR (CDCl3): δ 3.64 (s,
3H), 3.84 (s, 6H), 7.00 (s, 2H), 7.48 (m, 10H), 7.75 (m, 4H).
General Procedure E for the Synthesis of Compounds 9b, c,
g-o, s, and t. A stirred suspension of a thiophene derivative (one
of 13b and c, g-o, and s and t; 0.5 mmol) and hydrazine
monohydrate (29 µL, 0.6 mmol, 1.2 equiv) in abs EtOH (10 mL)
was refluxed for 3 h. The reaction mixture was then left at r.t. for
1 h, by which time the starting material was completely solubilized.
The solvent was evaporated, and the residue was partitioned
between EtOAc (10 mL) and water (5 mL). The separated organic
phase, which was washed with brine (2 mL) and dried, was
concentrated in vacuo to obtain a residue that was purified by
column chromatography using petroleum ether-EtOAc (7:3) as the
eluent to give the desired products 9b and c, g-o, or s and t.
(2-Amino-5-bromothiophen-3-yl)-(3,4,5-trimethoxyphenyl)-
methanone (9b). Yellow powder (67% yield); mp 119-120 °C;
1H NMR (CDCl3): δ 3.90 (m, 9H), 6.88 (s, 2H), 7.01 (s, 1H),
7.27 (bs, 2H). Anal. (C14H14BrNO4S): C, H, N.
2-[5-(4-Methylphenyl)-3-(3,4,5-trimethoxybenzoyl)-thiophen-
2-yl]-isoindole-1,3-dione (13j). The title compound was prepared
by general procedure D from 12a and p-methylphenylboronic acid.
EtOAc-petroleum ether (3:7) was used for flash chromatography.
Yellow solid (86% yield); mp 202-204 °C. 1H NMR (CDCl3): δ
2.38 (s, 3H), 3.69 (s, 3H), 3.82 (s, 6H), 7.00 (s, 2H), 7.24 (d, J )
9.2 Hz, 2H), 7.50 (d, J ) 9.2 Hz, 2H), 7.52 (s, 1H), 7.76 (m, 2H),
7.82 (m, 2H).
(2-Amino-5-iodothiophen-3-yl)-(3,4,5-trimethoxyphenyl)-meth-
anone (9c). Yellow powder (70% yield); mp 134-135 °C; 1H NMR
(CDCl3): δ 3.78 (s, 3H), 3.82 (s, 6H), 6.81 (s, 2H), 7.10 (s, 1H),
8.44 (bs, 2H). Anal. (C14H14INO4S): C, H, N.
[2-Amino-5-(4-chlorophenyl)-thiophen-3-yl]-(3,4,5-trimethoxy-
phenyl)-methanone (9g). Yellow powder (65% yield); mp 184-
2-[5-(4-Trifluoromethylphenyl)-3-(3,4,5-trimethoxybenzoyl)-
thiophen-2-yl]-isoindole-1,3-dione (13k). The title compound was
prepared by general procedure D from 12a and p-trifluorometh-
ylphenylboronic acid. EtOAc-petroleum ether (3:7) was used for
flash chromatography. Yellow solid (72% yield); mp 206-207 °C.
1H NMR (CDCl3): δ 3.70 (s, 3H), 3.83 (s, 6H), 7.01 (s, 2H), 7.65
(s, 1H), 7.70 (d, J ) 4.2 Hz, 2H), 7.75 (d, J ) 4.2 Hz, 2H), 7.83
(m, 2H), 7.85 (m, 2H).
2-[5-(4-Methoxyphenyl)-3-(3,4,5-trimethoxybenzoyl)-thiophen-
2-yl]-isoindole-1,3-dione (13l). The title compound was prepared
by procedure D from 12a and p-methoxyphenylboronic acid.
EtOAc-petroleum ether (3:7) was used for flash chromatography.
Yellow solid (82% yield); mp 181-182 °C. 1H NMR (CDCl3): δ
3.69 (s, 3H), 3.85 (s, 9H), 6.93 (s, 2H), 6.98 (d, J ) 9.2 Hz, 2H),
7.45 (s, 1H), 7.57 (d, J ) 9.2 Hz, 2H), 7.37 (m, 2H), 7.84 (m, 2H).
2-[5-(3-Methoxyphenyl)-3-(3,4,5-trimethoxybenzoyl)-thiophen-
2-yl]-isoindole-1,3-dione (13m). The title compound was prepared
by general procedure D from 12a and m-methoxyphenylboronic
acid. EtOAc-petroleum ether (3:7) was used for flash chromatog-
raphy. Yellow solid (75% yield); mp 202-204 °C. 1H NMR
(CDCl3): δ 3.69 (s, 3H), 3.86 (s, 9H), 6.98 (m, 1H), 7.00 (s, 2H),
7.05 (m, 1H), 7.12 (m, 1H), 7.34 (m, 1H), 7.54 (s, 1H), 7.76 (m,
2H), 7.83 (m, 2H).
1
186 °C; H NMR (CDCl3): δ 3.83 (s, 6H), 3.86 (s, 3H), 6.89 (s,
2H), 6.97 (bs, 2H), 7.09 (s, 1H), 7.20 (m, 4H). Anal. (C20H18-
ClNO4S): C, H, N.
[2-Amino-5-(3,4-dichlorophenyl)-thiophen-3-yl]-(3,4,5-tri-
methoxyphenyl)-methanone (9h). Yellow powder (55% yield);
mp 159-161 °C; 1H NMR (CDCl3): δ 3.83 (s, 6H), 3.86 (s, 3H),
6.87 (s, 2H), 6.92 (bs, 2H), 7.11 (s, 1H), 7.18 (s, 1H), 7.36 (m,
2H). Anal. (C20H17Cl2NO4S): C, H, N.
[2-Amino-5-(4-fluorophenyl)-thiophen-3-yl]-(3,4,5-trimethoxy-
phenyl)-methanone (9i). Yellow powder (78% yield); mp 174-
1
175 °C; H NMR (CDCl3): δ 3.90 (s, 6H), 3.92 (s, 3H), 6.95 (s,
2H), 7.02 (bs, 2H), 7.05 (m, 2H), 7.09 (s, 1H), 7.34 (m, 2H). Anal.
(C20H18FNO4S): C, H, N.
[2-Amino-5-(4-methylphenyl)-thiophen-3-yl]-(3,4,5-trimethoxy-
phenyl)-methanone (9j). Yellow powder (80% yield); mp 184-
1
185 °C; H NMR (CDCl3): δ 2.33 (s, 3H), 3.89 (s, 3H), 3.92 (s,
6H), 6.97 (s, 2H), 6.99 (bs, 2H), 7.12 (d, J ) 8.4 Hz, 2H), 7.13 (s,
1H), 7.28 (d, J ) 8.4 Hz, 2H). Anal. (C21H21NO4S): C, H, N.
[2-Amino-5-(4-trifluoromethylphenyl)-thiophen-3-yl]-(3,4,5-
trimethoxyphenyl)-methanone (9k). Yellow powder (70% yield);
mp 199-200 °C; 1H NMR (CDCl3): δ 3.90 (s, 6H), 3.93 (s, 3H),
6.96 (s, 2H), 7.09 (bs, 2H), 7.29 (s, 1H), 7.45 (d, J ) 8.6 Hz, 2H),
7,56 (d, J ) 8.6 Hz, 2H). Anal. (C21H18F3NO4S): C, H, N.
[2-Amino-5-(4-methoxyphenyl)-thiophen-3-yl]-(3,4,5-tri-meth-
oxyphenyl)-methanone (9l). Yellow powder (84% yield); mp 115-
2-[5-(2-Methoxyphenyl)-3-(3,4,5-trimethoxybenzoyl)-thiophen-
2-yl]-isoindole-1,3-dione (13n). The title compound was prepared
by general procedure D from 12a and o-methoxyphenylboronic acid.
EtOAc-petroleum ether (3:7) was used for flash chromatography.

3914 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Romagnoli et al.
1
116 °C; H NMR (CDCl3): δ 3.81 (s, 3H), 3.90 (s, 6H), 3.92 (s,
their number calculated as a percentage of the total number of cells
evaluated in three independent experiments. We previously dem-
onstrated that the employed concentrations of DMSO (used as
vehicle) did not alter the cell cycle parameters of treated cells.
Molecular Modeling. The crystal structure of DAMA-colchicine,
as deposited in the Protein Data Bank (pdb ID: ISA0),27 was used
as the reference structure. The 3D model of 9i molecule was built
by assembling the fragments from the SYBYL 7.0 software package
standard library (Tripos, St. Louis, MO). The resulting geometry
was optimized by semiempirical molecular orbital calculations using
the AM1 Hamiltonian35 (module MOPAC implemented in SYBYL).
The docking studies were carried out by means of the AutoDock
suite of software28 exploiting the 3D X-ray crystallography structure
of the DAMA-colchicine-tubulin complex. The flexible docking
simulations were performed using LGA. The 3D grids describing
the interactions of the atom types of the molecules under investiga-
tion (DAMA-colchicine and 9i) were precomputed using the
autogrid facility. In all the calculations, the energy scoring grids
(91 × 125 × 105 points, spacing 0.286 Å) were centered on the
geometric center of the inhibitor’s coordinates. The compounds were
subjected to 100 runs of the AutoDock search in which the default
values of the other parameters were used.
3H), 6.87 (d, J ) 8.8 Hz, 2H), 6.97 (s, 2H), 6.98 (bs, 2H), 7.04 (s,
1H), 7.32 (d, J ) 8.8 Hz, 2H). Anal. (C21H21NO5S): C, H, N.
[2-Amino-5-(3-methoxyphenyl)-thiophen-3-yl]-(3,4,5-tri-meth-
oxyphenyl)-methanone (9m). Yellow powder (76% yield); mp
160-161 °C; 1H NMR (CDCl3): δ 3.81 (s, 3H), 3.90 (s, 6H), 3.92
(s, 3H), 6.75 (m, 1H), 6.91 (m, 1H), 6.96 (s, 2H), 7.01 (bs, 2H),
7.18 (s, 1H), 7.20 (m, 2H). Anal. (C21H21NO5S): C, H, N.
[2-Amino-5-(2-methoxyphenyl)-thiophen-3-yl]-(3,4,5-trimeth-
oxyphenyl)-methanone (9n). Yellow powder (72% yield); mp 64-
1
66 °C; H NMR (CDCl3): δ 3.82 (s, 3H), 3.90 (s, 6H), 3.93 (s,
3H), 6.98 (s, 2H), 6.99 (bs, 2H), 7.08 (s, 1H), 7.13 (d, J ) 8.4 Hz,
1H), 7.32 (m, 2H), 7.34 (d, J ) 8.6 Hz, 1H). Anal. (C21H21NO5S):
C, H, N.
[2-Amino-5-(3,4,5-trimethoxyphenyl)-thiophen-3-yl]-(3,4,5-tri-
methoxyphenyl)-methanone (9o). Yellow powder (80% yield); mp
153-154 °C; 1H NMR (CDCl3): δ 3.84 (s, 3H), 3.87 (s, 6H), 3.90
(s, 6H), 3.93 (s, 3H), 6.59 (s, 2H), 6.98 (m, 2H), 6.99 (s, 2H), 7.06
(s, 1H). Anal. (C23H25NO7S): C, H, N.
(2-Amino-4-methyl-5-phenylthiophen-3-yl)-(3,4,5-trimeth-ox-
yphenyl)-methanone (9s). Yellow powder (75% yield); mp 130-
131 °C; 1H NMR (CDCl3): δ 1.83 (s, 3H), 3.89 (s, 9H), 6.40 (bs,
2H), 6.87 (s, 2H), 7.36 (m, 5H). Anal. (C21H21NO4S): C, H, N.
(2-Amino-4,5-diphenylthiophen-3-yl)-(3,4,5-trimethoxyphenyl)-
methanone (9t). Yellow powder (61% yield); mp 153-155 °C;
1H NMR (CDCl3): δ 3.70 (s, 9H), 6.60 (s, 2H), 6.86 (bs, 2H),
7.02 (m, 5H), 7.18 (m, 5H). Anal. (C26H23NO4S): C, H, N.
Inhibition of the Growth of Murine L1210 and Human K562
Cells. The murine lymphocytic L1210 and the human chronic
myelogenous K562 leukemia cell lines were obtained from the
American Type Culture Collection. All test compounds were
dissolved in dimethyl sulfoxide (DMSO) at 1 mg/mL immediately
before use and diluted in the medium before addition to the cells.
Both cell lines were cultured in an RPMI 1640 medium (GIBCO)
supplemented with 10% bovine fetal calf serum (Flow, Irvine,
U.K.), 2 mM L-glutamine (GIBCO), 10 mM â-mercaptoethanol,
100 U/mL of penicillin, and 100 µg/mL of streptomycin. To
determine the effects of the studied compounds on cell growth,
exponentially growing L1210 and K562 cells were exposed to
increasing concentrations of drugs, and the cell number/mL value
was determined after 48 h using a model ZBI Coulter Counter
(Coulter Electronics, Hialeah, FL). The results were expressed as
IC50 values (concentration causing 50% inhibition relative to
untreated controls). All experiments were repeated at least three
times. For each drug concentration, duplicate cultures were used.
Considering the possible antiproliferative effects of DMSO, control
cultures were always performed using the maximum levels of
DMSO employed for the administration of tested compounds. The
concentration of DMSO in the array was never higher than 0.01%
and did not affect the cell growth of the employed cell lines.
Effects on Tubulin Polymerization and Colchicine Binding
to Tubulin. Bovine brain tubulin was purified as previously
described.31 To evaluate the effect of the compounds on tubulin
assembly in vitro,22a the compounds were preincubated across a
concentration range with 10 µM tubulin in a glutamate buffer at
30 °C and then cooled to 0 °C. After the addition of GTP, the
mixtures were warmed to 30°C, and the assembly of tubulin was
observed turbidimetrically. The IC50 value was defined as the
compound concentration that inhibited the extent of assembly by
50%. The capacity of the test compounds to inhibit colchicine
binding to tubulin was measured as described,22b except that the
reaction mixtures contained 1 µM tubulin, 5 µM [3H]-colchicine,
and 1 µM test compound.
Acknowledgment. R.G. thanks AIRC and UE Obiettivo 2
for grant support. This work was partially supported by National
Cancer Institute Contract No. NO-CO-12400. We thank Drs.
Leonardo Vizziello and Martina Baruffa for performing cell
cycle analyses and the measurements of apoptosis.
Supporting Information Available: Elemental analyses for
compounds 9a-v. This material is available free of charge via the
Internet at http://pubs.acs.org.
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