Synthesis of novel antimitotic agents based on 2-amino-3-aroyl-5-(hetero) arylethynyl thiophene derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.11.083

Microtubules are dynamic structures that play a crucial role in cellular division and are recognized as an important target for cancer therapy. In search of new compounds with strong antiproliferative activity and simple molecular structure, a new series of 2-amino-3-(3′,4′,5′- trimethoxybenzoyl)-5-(hetero)aryl ethynyl thiophene derivatives was prepared by the Sonogashira coupling reaction of the corresponding 5-bromothiophenes with several (hetero)aryl acetylenes. When these compounds were analyzed in vitro for their inhibition of cell proliferation, the 2- and 3-thiophenyl acetylene derivatives were the most powerful compounds, both of which exerted cytostatic effects at submicromolar concentrations. In contrast, the presence of a more flexible ethyl chain between the (hetero)aryl and the 5-position of the thiophene ring resulted in significant reduction in activity relative to the 5-(hetero)aryl acetylene substituted derivatives. The effects of a selected series of compounds on cell cycle progression correlated well with their strong antiproliferative activity and inhibition of tubulin polymerization. We found that the antiproliferative effects of the most active compounds were associated with increase of the proportion of cells in the G₂/M and sub-G ₁ phases of the cell cycle.

Full text of DOI:10.1016/j.bmcl.2010.11.083

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2746–2751
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel antimitotic agents based
on 2-amino-3-aroyl-5-(hetero)arylethynyl thiophene derivatives
Romeo Romagnoli a, , Pier Giovanni Baraldi a, , Olga Cruz-Lopez a, Manlio Tolomeo b, Antonietta Di Cristina b,
⇑
⇑
Rosaria Maria Pipitone b, Stefania Grimaudo b, Jan Balzarini c, Andrea Brancale d, Ernest Hamel e
a Dipartimento di Scienze Farmaceutiche, Università di Ferrara, 44100 Ferrara, Italy
b Centro Interdipartimentale di Ricerca in Oncologia Clinica e Dipartimento Biomedico di Medicina Interna e Specialistica, Università di Palermo, Palermo, Italy
c Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Minderbroedersstraat 10, B-3000 Leuven, Belgium
d The Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK
e Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Microtubules are dynamic structures that play a crucial role in cellular division and are recognized as an
important target for cancer therapy. In search of new compounds with strong antiproliferative activity
and simple molecular structure, a new series of 2-amino-3-(30,40,50-trimethoxybenzoyl)-5-(hetero)aryl
ethynyl thiophene derivatives was prepared by the Sonogashira coupling reaction of the corresponding
5-bromothiophenes with several (hetero)aryl acetylenes. When these compounds were analyzed
in vitro for their inhibition of cell proliferation, the 2- and 3-thiophenyl acetylene derivatives were the
most powerful compounds, both of which exerted cytostatic effects at submicromolar concentrations.
In contrast, the presence of a more flexible ethyl chain between the (hetero)aryl and the 5-position of
the thiophene ring resulted in significant reduction in activity relative to the 5-(hetero)aryl acetylene
substituted derivatives. The effects of a selected series of compounds on cell cycle progression correlated
well with their strong antiproliferative activity and inhibition of tubulin polymerization. We found that
the antiproliferative effects of the most active compounds were associated with increase of the propor-
tion of cells in the G2/M and sub-G1 phases of the cell cycle.
Received 15 October 2010
Revised 9 November 2010
Accepted 16 November 2010
Available online 21 November 2010
Keywords:
Thiophene
Inhibition of tubulin polymerization
Inhibition of tumor cell growth
Antiproliferative agents
Ó 2010 Elsevier Ltd. All rights reserved.
The microtubule system of eukaryotic cells is essential in a
variety of fundamental cellular processes, including mitosis,
formation and maintenance of cell shape, regulation of motility,
cell signaling, secretion, and intracellular transport.1,2 There has
long been considerable interest in the discovery and development
of novel small molecules, many of which are natural products, able
to inhibit tubulin polymerization for the treatment of cancer.3–5
Combretastatin A-4 (CA-4, 1), isolated from an African willow,
Combretum caffrum (Combretaceae), shows interesting anticancer
potential due to its antitubulin properties.6 Previous studies have
found that the 30,40,50-trimethoxy group on the A ring was essential
for strong activity, while the 30-hydroxy group on the B-ring is not
necessary for potent activity.7–9 CA-4 strongly binds to the
colchicine site of tubulin, and this binding prevents tubulin poly-
merization and causes an antimitotic effect. CA-4 inhibits cell
growth at low to mid nanomolar concentrations, and its structural
simplicity makes CA-4 of great interest from the medicinal chem-
istry viewpoint.10
For these reasons, a large number of synthetic CA-4 analogues
have been synthesized and evaluated in structure–activity relation-
ship (SAR) studies.7–12 Replacing the double bound of CA-4 with a
carbonyl group furnished a benzophenone-type CA-4 analogue
named phenstatin (2), which was found to be a very strong cytotoxic
agent with the same characteristics as CA-4.13 The 2-aminobenzo-
phenone derivative 3 showed significantly increased cytotoxicity
against many human cancer cell lines as compared with phenstatin
2, but 3 was nevertheless slightly less potent than CA-4.14
The classical bioisosteric equivalence between benzene and
thiophene prompted us to synthesize a series of 2-amino-3-
(30,40,50-trimethoxybenzoyl)thiophene derivatives with general
formula 4, in which a 2-aminothiophene system replaced the
2-aminobenzene moiety in the 2-aminophenstatin analogue 3.15
The 4,5-unsubstituted derivative 4a displayed modest activity,
which was little affected with alkyl or halogen substituents at
C-5. The weak activity of 4a was enhanced up to 12-fold by the
4-methyl substitution (compound 4b). The contribution of the
phenyl group to activity was position dependent. Placing a phenyl
⇑
Corresponding authors. Tel.: +39(0)532 455303; fax: +39(0)532 455953 (R.R.);
tel.: 39(0)532 291293; fax: +39(0)532 455953 (P.G.B.).
E-mail addresses: rmr@unife.it (R. Romagnoli), baraldi@unife.it (P.G. Baraldi).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.083

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2746–2751
2747
at C-4 (4c) resulted in an inactive compound, while the corre-
5-diol (the dimer of
a-mercaptoacetaldehyde) in ethanol with
sponding C-5 phenyl derivative 4d was a potent inhibitor of tubu-
lin polymerization and showed strong antiproliferative activities
against two leukemic cell lines, L1210 and K562, with accumula-
tion of cells in the G2/M phase of the cell cycle.
triethylamine (TEA) (2.2 equiv) followed by the addition of
3-(30,40,50-trimethoxyphenyl)-3-oxopropanenitrile 6,15 furnished
the corresponding 2-amino-3-(30,40,50-trimethoxybenzoyl)thio-
phenes 7a or 7b, respectively. These latter compounds were con-
verted almost quantitatively to the phthalimido derivatives 8a
and 8b using phthalic anhydride in acetic acid, and the subsequent
regioselective bromination of 8a and 8b in a mixture of acetic acid
and sodium acetate using bromine, furnished intermediates 5-bro-
mothiophene derivatives 9a and 9b. These latter compounds were
coupled by a standard Sonogashira cross-coupling reaction17 with
the appropriate alkyne in the presence of cuprous iodide (CuI),
bis(triphenylphosphine)-palladium chloride [PdCl2(PPh3)2] and a
mixture of TEA and DMF, to afford the (hetero)arylacetylenic deriv-
atives 10a–h. The removal of the N-protected phthaloyl group was
accomplished by the use of hydrazine in refluxing ethanol, to af-
ford compounds 5a–h. Partial hydrogenation of the triple C–C
bonds of 5e and 5g with Lindlar’s catalyst (5% palladium on cal-
cium carbonate poisoned with lead) gave 5i and 5j, respectively,
as (Z)-isomers in acceptable yields. Compounds 5k–o, character-
ized by the presence of a more flexible ethyl linker, were prepared
starting from derivatives 5a–d and 5g, respectively, by catalytic
hydrogenation of the triple bond over 10% palladium on charcoal
(Pd/C).
On the basis of previous encouraging results on the antitumor
activity of compound 4d, we have now extended this study by
the synthesis and biological evaluation of a related series of com-
pounds with general structure 5, characterized by a common
2-amino-3-(30,40,50-trimethoxybenzoyl)-thiophene core but with
an aryl/heteroaryl ethynedyl moiety at the C-5-position of the thi-
ophene ring. The substituents examined at the 4-position included
hydrogen or methyl. Partial or complete reduction of ethynyl-
linked compounds 5a–h afforded vinyl- and ethyl-linked deriva-
tives 5i–j and 5k–o, respectively. By the preparation of compounds
5c, 5d, 5m and 5n, we explored the SAR by examining electron-
releasing (Me and MeO) groups on the p-position of the phenyl
of phenyethynyl or phenylethyl moieties (compounds 5c, 5d, and
5m, 5n, respectively) (Chart 1).
The 30,40,50-trimethoxyphenyl of the 2-benzoyl moiety was kept
unchanged because it is the characteristic structural requirement
for activity in a large series of inhibitors of tubulin polymerization,
such as colchicine, combretastatin A-4, and podophyllotoxin.7,16
The approach taken for the preparation of compounds 5a–o is
shown in Scheme 1. The treatment of the 2,5-dimethyl-(1,4)dithi-
Table 1 summarizes the growth inhibitory effects of derivatives
5a–o against murine leukemia (L1210), human cervix carcinoma
ane-2,5-diol (the dimer of
a-mercaptoacetone) or 1,4-dithiane-2,
R1
H3CO
H3CO
O
H3CO
H3CO
R2
R3
A
B
CH3O
OH
CH3O
OCH3
R1=H, R2=OH, R3=OCH3, Phenstatin, 2
R1=NH2, R2=H, R3=OCH3, 3
Combretastatin A-4 (CA-4), 1
O
NH2
OCH3
O
H3CO
S
R2
R1
OCH3
H3CO
CH3O
R1
OCH3
4
NH2
S
R1=R2=H, 4a
R2
5a-h
R1=Me, R2=H, 4b
R1=Ph, R2=H, 4c
R1=H, R2=Ph, 4d
R1=Me, R2=Ph, 4e
R1=H, R2=C6H5, 5a
R1=CH3, R2=C6H5, 5b
R1=H, R2=p-Me-C6H4, 5c
R1=H, R2=p-OMe-C6H4, 5d
R1=H, R2=thiophen-2-yl, 5e
R1=Me, R2=thiophen-2-yl, 5f
R1=H, R2=thiophen-3-yl, 5g
R1=Me, R2=thiophen-3-yl, 5h
OCH3
OCH3
OCH3
O
R1
R2
NH2
S
5k-o
OCH3
O
R1=H, R2=C6H5, 5k
OCH3
R1=CH3, R2=C6H5, 5l
R1=H, R2=p-Me-C6H4, 5m
R1=H, R2=p-OMe-C6H4, 5n
R1=H, R2=thiophen-3-yl, 5o
OCH3
R
NH2
S
5i-j
R=thiophen-2-yl, 5i
R=thiophen-3-yl, 5j
Chart 1.

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R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2746–2751
H3CO
H3CO
R1
OCH3
H3CO
OCH3
H3CO
R1
H3CO
H3CO
H3CO
O
b
a
O
O
O
R1=H, 8a
CN
R1=Me, 8b
6
N
NH2
S
S
8ab
7ab
O
R1=H, 7a
R1=Me, 7b
c
H3CO
OCH3
H3CO
R1
Br
R1=H, 9a
O
R1=H, R2=C6H5, 5k
R1=CH3, R2=C6H5, 5l
R1=Me, 9b
OCH3
OCH3
OCH3
O
O
N
R1=H, R2=p-Me-C6H4, 5m
R1=H, R2=p-OMe-C6H4, 5n
R1=H, R2=thiophen-3-yl, 5o
S
9ab
R1
O
R2
NH2
S
5k-o
d
f
OCH3
H3CO
H3CO
R1
OCH3
H3CO
H3CO
R1
OCH3
O
O
O
OCH3
e
g
O
OCH3
R
N
S
NH2
S
R2
5i-j
NH2
10a-h
S
O
R2
5a-h
R=thiophen-2-yl, 5i
R=thiophen-3-yl, 5j
R1=H, R2=C6H5, 10a, 5a
R1=CH3, R2=C6H5, 10b, 5b
R1=H, R2=p-Me-C6H4, 10b, 5c
R1=H, R2=p-OMe-C6H4, 10d, 5d
R1=H, R2=thiophen-2-yl, 10e, 5e
R1=Me, R2=thiophen-2-yl, 10f, 5f
R1=H, R2=thiophen-3-yl,10g, 5g
R1=Me, R2=thiophen-3-yl, 10h, 5h
Scheme 1. Reagents: (a) 2,5-dimethyl-(1,4)dithiane-2,5-diol or 1,4-dithiane-2,5-diol, TEA, EtOH; (b) phthalic anhydride, AcOH; (c) Br2, AcOH, AcONa, rt;
(d) (hetero)arylacetylene, PdCl2(PPh3)2, CuI, TEA–DMF (1:1, v/v), 80 °C; (e) NH2NH2, EtOH; (f) H2, 10% Pd/C, EtOH (for 5a–d and 5g); (g) H2, Lindlar’s catalyst, EtOH (for 5e
and 5g).
(HeLa), murine mammary carcinoma (FM3A) and human
T-lymphoblastoid (Molt/4 and CEM) cells. The alkylating agent
melphalan and compounds 4de were used as reference drugs. 2-
Amino-3-(30,40,50-trimethoxybenzoyl)-5-(thiophen-30-yl ethynyl)
thiophene 5g possessed the highest potency and inhibited the
growth of L1210, FM3A, Molt/4, CEM, and HeLa cancer cell lines with
introduction of an electron-releasing methyl or methoxy group at
the para-position of the phenylethynyl unit (analogues 5c and
5d, respectively) caused substantial loss of activity (IC50 >10 lM).
The introduction of a methyl group in the 4-position of the 2-ami-
no-3-(30,40,50-trimethoxybenzoyl)-thiophene skeleton (compound
5b) led to no change in activity, relative to 5a, against the tumor
cells.
IC50’s of 0.21, 0.13, 0.096, 0.11, and 0.20 lM, respectively. The anti-
proliferative potency of 5a was diminished relative to compound 4d
with the phenyl directly bounded at the C5 position of the 2-amino-
3-(30,40,50-trimethoxybenzoyl)-thiophene core. Derivatives 5ab and
5e–j were found to be significantly more active than melphalan
against the tested cancer cell lines. The presence of an ethynyl
(compounds 5a–h) or ethenyl (compounds 5i and 5j) group at the
5-position of 2-amino-3-(30,40,50-trimethoxybenzoyl)thiophene is
essential for activity, sice congeners with an ethyl group at that
position (compounds 5k and 5m–o) were generally inactive.
While the unsubstituted phenylacetylene derivative 5a exerted
antiproliferative activity at low micromolar concentrations, the
The bioisosteric replacement of phenyl in 5a by a thiophene, in
either regioisomeric orientation, to furnish the 20- and 30-thiophenyl
derivatives 5e and 5g, have a beneficial effect on antiproliferative
activity. In contrast to the situation with thiophen-20-ylacetylene
derivatives 5e and 5f, where the introduction of methyl group in
the 4-position of the 2-amino-3-(30,40,50-trimethoxybenzoyl)-thio-
phene skeleton (compound 5f) led to little change in activity, with
the isomeric thiophen-30-ylacetylene derivative 5g the 4-methyl
group (compound 5h) was not well-tolerated and caused a two to
fourfold reduction in activity against L1210, FM3A, Molt4, and
CEM cells, but no effect on activity against HeLa cells.

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2746–2751
2749
Table 1
In vitro inhibitory effects of compounds 4d, 4e, 5a–o and melphalan on proliferation of murine leukemia (L1210), murine mammary carcinoma (FM3A), human cervix carcinoma
(HeLa) and human T-lymphocyte (Molt/4 and CEM) cells
Compound
IC50 S.D (
Molt4/C8
l
M)a
L1210
FM3A/0
CEM/0
HeLa
5a
5b
5c
2.6 0.1
3.0 0.6
>10
2.5 0.1
3.3 0.9
>10
1.5 0.4
2.1 0.0
>10
1.9 0.0
2.1 0.1
>10
1.6 0.7
1.6 0.6
>10
5d
>10
>10
>10
>10
>10
5e
5f
5g
5h
5i
5j
5k
5l
0.52 0.01
0.52 0.03
0.21 0.04
0.45 0.01
0.16 0.13
2.3 0.0
>10
>10
>10
>10
0.42 0.01
0.46 0.00
0.13 0.00
0.33 0.04
0.41 0.39
0.41 0.01
0.42 0.06
0.47 0.06
0.46 0.04
0.53 0.00
0.11 0.01
0.48 0.00
0.56 0.61
1.3 1.1
8.7 0.0
>10
0.20 0.02
0.18 0.01
0.20 0.12
0.12 0.04
0.16 0.01
1.3 0.7
7.3 3.0
>10
0.096 0.012
0.45 0.03
0.13 0.20
0.63 0.21
6.6 1.6
>10
11
2
>10
>10
>10
5m
5n
>10
>10
>10
>10
>10
>10
5o
4d
4e
5.0 3.3
0.012 0.00
1.3 1.1
8.6 0.3
5.2 4.0
0.0096 0.00
1.6 0.6
1.9 0.4
0.0030 0.00
0.31 0.09
n.d.
2.1 0.2
0.0029 0.00
0.16 0.02
3.5 0.1
7.6 0.2
0.012 0.00
0.85 0.44
1.9 0.1
Melphalan
3.6 0.3
n.d. = not determined.
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the IC50 SD from the dose–response curves of at least three
independent experiments.
For the thiophen-20-yl derivatives 5e–i, the activity of acetylene
derivative 5e was inferior to that of the partially reduced (Z)-ethyl-
ene analogue 5i only against L1210 and Molt4 cells.
The order of inhibitory action on tubulin assembly was 5h > 5f >
5e = 5g = CA4 > 5i > 4d, which was not consistent with the results
of the antiproliferative assays. (5g had the greatest antiprolifera-
tive activity, although the differences between the most active
compounds in the new series were not great). The reduced antipro-
liferative activities of 5h and 5f as compared with 4d may result
from poor permeability into cells, poor solubility in the tissue
culture medium or any other mechanism limiting the accessibility
of these molecules to cellular tubulin. Nevertheless, we could not
exclude the possibility that 4d may affect other molecular targets
in addition to microtubules resulting in the enhanced antiprolifer-
ative activity.
In the series of isomeric thiophen-30-yl derivatives 5g, 5j, and
5o, compound 5g, with a rigid ethynyl spacer between the C-5 po-
sition of the 2-amino-3-(30,40,50-trimethoxybenzoyl)-thiophene
skeleton and the thiophen-30-yl moiety, was more potent as an
antiproliferative agent than the corresponding ethenyl derivative
5j and the ethyl group (analogue 5o) led to a further reduction in
activity.
To investigate whether the antiproliferative activities of these
compounds were related to an interaction with tubulin, we exam-
ined the effects of the more active compounds 5e–i on the
polymerization of purified tubulin,18 in comparison with the
potent antimitotic compounds 4d and CA-4 (Table 2). The same
compounds were also examined for inhibitory effects on the bind-
ing of [3H]colchicine to tubulin.19
Compounds 5e–i all strongly inhibited tubulin assembly, and
compounds 5f and 5h seemed to be even more active than the
reference compound CA-4. 5h was found to be the most active
In the colchicine binding studies, compounds 5e–i potently
inhibited the binding of [3H]colchicine to tubulin, since 70–90%
inhibition occurred with these agents and colchicine both at
5 lM. None, however, was quite as potent as CA-4, which in these
experiments inhibited colchicine binding by 99%. CA-4 proved also
to be somewhat more potent than compound 5h, which was the
most potent of the new agents, but otherwise the order of activity
in the two tubulin-based assays was identical.
(IC50 = 0.62
l
M) in the in vitro tubulin polymerization assay,
The results are consistent with the conclusion that the antipro-
liferative activity of these compounds derives from an interaction
with the colchicine site of tubulin and interference with microtu-
bule assembly.
having almost twice the potency of CA-4. In comparison with 4d,
compound 5h was three times as potent as an inhibitor of tubulin
assembly, but it was much less active as an antiproliferative agent.
To further support this observation, we have performed a series
of molecular docking simulation using a procedure reported previ-
ously.20 Interestingly, the ethynyl analogues 5f–h showed some
peculiar features in their binding mode (Fig. 1). The trimethoxy-
phenyl group is placed in a similar position as the corresponding
ring of the co-crystallized DAMA-colchicine, and the amino group
formed a hydrogen bond with Thr179. At the same time, the ethy-
nyl side chain reaches a small hydrophobic cleft formed by Ala354
and Leu248, outside the colchicine binging pocket. Interestingly,
the methyl group on the amino-thiophene ring of 5h is also in con-
tact with Leu248, and this interaction could explain the increased
antitubulin activity of 5h versus 5g, which lacks this substituent.
These results are in accord with the experimental data.
Table 2
Inhibition of tubulin polymerization and colchicine binding by compounds 5e–i, 4d
and CA-4
Compound
Tubulin assemblya
IC50 S.D (lM)
Colchicine bindingb
%
S.D
5
lM drug
1 lM drug
5e
5f
5g
5h
5i
4d
CA-4 (1)
1.0 0.1
0.84 0.1
1.0 0.0
0.62 0.1
1.2 0.1
1.9 0.1
1.0 0.2
70
81
76
90
73
2
3
4
2
3
n.d.
47
40
60
n.d.
60
4
2
4
n.d.
5
3
99 0.3
90
n.d. = not determined.
Because molecules exhibiting effects on tubulin assembly
should cause alteration of cell cycle parameters with preferential
G2–M blockade, flow cytometry analysis was performed to deter-
a
Inhibition of tubulin polymerization. Tubulin was at 10
l
M.
Inhibition of [3H]colchicine binding. Tubulin and colchicine were at 1 and 5
concentration, respectively.
b
lM

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R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2746–2751
Figure 1. Proposed binding mode of 5g (in green) and 5h (in purple). DAMA-colchicine is represented in orange.
Table 3
Effects of compounds 5e–i on K562 cell cycle progression
triple bond. It is clear that the substitution pattern on the 5-posi-
tion of thiophene ring play an important role for antiproliferative
activities, with the thiophen-30-yl acetylene group as the best sub-
stituent of those examined. In the series of thiophen-30-yl deriva-
tives 5g, 5j, and 5o, the cell growth inhibition effect increased
with increasing rigidity of the linker at the C-5 position of the thio-
phen-30-yl group in the 2-amino-3-(30,40,50-trimethoxybenzoyl)-
thiophene skeleton. The presence of an acetylene spacer is optimal
for activity, while the reduction of acetylene to ethyl decreased
antiproliferative activity by 10–100-fold against all cancer cell
lines.
The biological evaluation included extensive antiproliferative
studies with five cancer cell lines and examination of some of
the most active compounds on tubulin assembly and disruption
of the cell cycle. In the latter studies, we observed accumulation
of G2–M and apoptotic cells, effects expected for compounds that
interact with tubulin. 2-Amino-3-(30,40,50-trimethoxybenzoyl)-5-
(thiophen-30-yl ethynyl) thiophene 5g was the most active of the
new compounds against cell growth, with IC50 values in the range
a
Compound
IC100 (nM)
Cell cycle distribution (%)
b
Sub-G1
G0G1
S
G2/M
Control
5e
5f
5g
5h
2.2
16.9
6.00
15.3
16.4
16.6
43.40
22.8
3.72
21.9
25.4
21.0
40.30
25.0
13.8
40.1
25.6
41.2
16.3
52.3
82.5
38.0
49.0
37.9
800 12
500 10
600 14
900 18
500 15
5i
a
Compound concentration required to inhibit tumor cell proliferation by 100%.
Data are expressed as the mean SD from the dose–response curves of at least
three independent experiments.
b
Percentage of the cell population with hypodiploid DNA content peak (apop-
totic cells).
mine the effect of compounds 5e–i on K562 (human chronic
myelogenous leukemia) cells. The K562 cell line was used because
of its rapid proliferation and high sensitivity to standard anticancer
agents and in order to determine whether these compounds had
activity against human transformed cells.21 Cells were cultured
for 24 h in the presence of each compound at the concentration
able to inhibit 100% cell growth (IC100) after 24 h, and the results
are summarized in Table 3. IC100 was the concentration able to
maintain the number of living cells after 24 h (T = 24 h) at
200,000/mL which is the number of cells in the control at T = 0
or the number of cells plated before the treatment. This concentra-
tion induced a low percentage of apoptosis (from 6% for compound
5f to 16.9% for compound 5e, as shown by sub-G1 peaks in Table 3)
but caused important and evident modifications in cell cycle distri-
bution. In fact, all tested compounds caused an increase in the pro-
portion of cells in the G2–M peak relative to the untreated control.
In conclusion, a series of analogues with an ethynyl, ethylene or
ethyl chain at the C-5 position of 2-amino-3-(30,40,50-trimethoxy-
benzoyl)-thiophene core were produced by Sonogashira coupling
of the corresponding 5-bromo derivatives with aryl/heteroaryl al-
kynes, followed by partial or complete reduction of the ethynedyl
of 0.1–0.2 lM. The potency of 5g as an inhibitor of tubulin assem-
bly was comparable to that of CA-4. When the thiophen-30-yl
group was replaced by a phenyl moiety, activity dropped dramat-
ically. Molecular modeling studies were also performed, and the
proposed binding mode for compound 5h in the colchicine site of
tubulin was consistent with the experimental data.
Acknowledgments
The authors would like to thank Dr. Alberto Casolari and
Dr. Erika Marzola for the technical assistance.
Supplementary data
Supplementary data (detailed biological protocols, synthesis
and spectroscopic data for compounds 5a–o and 10a–h) associated
with this article can be found, in the online version, at doi:10.1016/
j.bmcl.2010.11.083.

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2746–2751
2751
12. Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. J. Med.
Chem. 2006, 49, 3033.
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