Design, synthesis, and biological evaluation of thiophene analogues of chalcones

Article abstract of DOI:10.1016/j.bmc.2008.04.026

Chalcones are characterized by possessing an enone moiety between two aromatic rings. A series of chalcone-like agents, in which the double bond of the enone system is embedded within a thiophene ring, were synthesized and evaluated for antiproliferative

Full text of DOI:10.1016/j.bmc.2008.04.026

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5367–5376
Design, synthesis, and biological evaluation of thiophene
analogues of chalcones
a,
a
Romeo Romagnoli,a,* Pier Giovanni Baraldi, Maria Dora Carrion,
*
Carlota Lopez Cara,a Olga Cruz-Lopez,a Delia Preti,a Manlio Tolomeo,b
Stefania Grimaudo,b Antonella Di Cristina,b Nicola Zonta,c Jan Balzarini,d
Andrea Brancale,c Taradas Sarkare and Ernest Hamele
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy
bDivisione di Ematologia e Servizio AIDS, Dipartimento di Oncologia, Policlinico ‘‘P. Giaccone’’,
`
Universita di Palermo, Palermo, Italy
cThe Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10, UK
dRega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Minderbroedersstraat 10, B-3000 Leuven, Belgium
eToxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA
Received 21 February 2008; revised 5 April 2008; accepted 11 April 2008
Available online 15 April 2008
Abstract—Chalcones are characterized by possessing an enone moiety between two aromatic rings. A series of chalcone-like agents,
in which the double bond of the enone system is embedded within a thiophene ring, were synthesized and evaluated for antiprolif-
erative activity and inhibition of tubulin assembly and colchicine binding to tubulin. The replacement of the double bond with a
thiophene maintains antiproliferative activity and therefore must not significantly alter the relative conformation of the two aryl
rings. The synthesized compounds were found to inhibit the growth of several cancer cell lines at nanomolar to low micromolar
concentrations. In general, all compounds having significant antiproliferative activity inhibited tubulin polymerization with an
IC50 < 2 lM. Several of these compounds caused K562 cells to arrest in the G2/M phase of the cell cycle.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Numerous chemically diverse antimitotic agents, many
of which are derived from natural products, have been
found to interact specifically with tubulin.3 Chalcones
with general structure 1 (Chart 1) are known to exhi-
bit antimitotic properties caused by the inhibition of
tubulin polymerization.4 Chemically, they are open-
chained molecules consisting of two aromatic rings
linked by a three-carbon enone fragment. Several re-
search groups have shown that the s-cis conformation
of chalcones is important for their biological activity.5
Their simple structure and ease of preparation make
chalcones an attractive scaffold for structure–activity
relationship (SAR) studies, and a wide number of
substituted chalcones have been synthesized to evalu-
ate the effects of various functional groups on biolog-
ical activity.4
The microtubule system of eukaryotic cells plays impor-
tant roles in regulating cell architecture, and has an
essential role in cell division, since microtubules are a
key component of the mitotic spindle.1 Microtubules
are a dynamic cellular compartment in both neoplastic
and normal cells. This dynamicity is characterized by
the continuous turnover of a,b-tubulin heterodimers in
the polymeric microtubules. They are involved in a vari-
ety of fundamental cellular processes, such as regulation
of motility, cell signalling, formation and maintenance
of cell shape, as well as transport of material within
the cell.2
Keywords: Chalcones; Tubulin; Colchicine.
It has been reported that hydrogenation or bromination
across the carbon–carbon double bond or its transfor-
mation into the corresponding epoxide dramatically
*
Corresponding author. Tel.: +39 0532 455303; fax: +39 0532 455953
(R.R.); tel.: +39 0532 455293; fax: +39 0532 455953 (P.G.B.); e-mail
addresses: rmr@unife.it; baraldi@unife.it
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.026

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R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 5367–5376
OCH3
OCH3
OCH3
OCH3
R
OCH3
R
OCH3
S
O
O
2a-p
1a-g
2a, R=C6H5
1a, R=C6H5
2b, R=2-thienyl
2c, R=3-thienyl
2d, R=p-F-C6H4
1b, R=p-F-C6H4
1c, R=p-Cl-C6H4
1d, R=p-OCH3-C6H4
1e, R=m-OCH3-C6H4
1f, R=o-OCH3-C6H4
1g, R=m,p-2(OCH3)-C6H3
2e, R=m-F-C6H4
2f, R=o-F-C6H4
2g, R=p-Cl-C6H4
Figure 1. Structure alignment between 1f (in magenta) and 2m (in
grey).
2h, R=o-Cl-C6H4
2i, R=p-CH3-C6H4
2j, R=o-CH3-C6H4
2k, R=p-OCH3-C6H4
2l, R=m-OCH3-C6H4
2m, R=o-OCH3-C6H4
2n, R=m,p-2(OCH3)-C6H3
2o, R=p-NO2-C6H4
2p, R=m-NO2-C6H4
2. Chemistry
2-(30,40,50-Trimethoxybenzoyl)-5-arylthiophene deriva-
tives with general structure 2 were synthesized by a
two-step procedure, as shown in Scheme 1. The first step
consisted of the synthesis of b-chlorocinnamaldehydes
4a–p , which in turn were obtained in good yields (40–
80%) by Vilsmeier–Haack chloroformylation applied to
commercially available acetophenones 3a–p , treated with
N,N-dimethylformamide and phosphorus oxychloride.11
The ‘one-pot’ cyclization in refluxing ethanol of 4a–p with
the a-mercapto ketone anion 7, generated in situ by
treating O-ethyl-S-[2-oxo-2-(3,4,5-trimethoxyphenyl)-
ethyl]dithiocarbonate 612 with piperidine, produced the
2-(30,40,50-trimethoxybenzoyl)-5-arylthiophene deriva-
tives 2a–p in moderate to good yields (40–60%).
Chart 1. General structure of chalcones 1a–g and thiophene deriva-
tives 2a–p.
reduced the chalcone activity.6 In addition, heterocyclic
derivatives of chalcones with the enone system replaced
with a pyrazole7 or pyrazoline8 ring have been systemat-
ically investigated, and these analogues had a reduced
activity when compared with the corresponding chal-
cone derivatives. These results demonstrated that the
relative positions of the two aryl rings in the molecule
play an essential role in the activity of chalcones and
that the double bond seems to be a crucial moiety for
biological activity. However, the clinical development
of chalcones is limited by their chemical instability in
vivo. The a,b-unsaturated carbonyl system in these com-
pounds can undergo Michael reactions with biological
nucleophiles, hence reducing their reliability as selective
tubulin inhibitors.9
3. Results and discussion
Table 1 summarizes the antiproliferative effects of thio-
phene derivatives 2a–p against a panel of tumour cell
lines using chalcones 1a–g13 as reference compounds.
Several derivatives demonstrated substantial growth
inhibitory effects against murine leukaemia (L1210),
murine mammary carcinoma (FM3A), human T-lym-
phoblastoid (Molt/4 and CEM) and human cervix carci-
noma (HeLa) cells. In general, the antiproliferative
activities of the tested compounds were more pro-
nounced against Molt/4 and HeLa cells. Of the tested
compounds, derivatives 2abdfimn possessed the highest
overall potency, with IC50 values of 0.2–1.4 lM against
the five cell lines. In general, the thiophene derivatives
2adglmn are more active than their chalcone analogues
with general structure 1. In particular, the methoxy
group at the 20-position of the phenyl ring enhanced
antiproliferative activity.
In this paper, we report a series of chalcone-like agents
with general structure 2, characterized by the presence
of a 2-(3,4,5-trimethoxybenzoyl)-5-aryl/heteroaryl-thio-
phene nucleus. The trimethoxyphenyl ring was thought
to be indispensable for retaining potency in the series
of molecules which occupy the colchicine binding
site.7,8,10
These new derivatives were developed with the aim to
determine whether the replacement of the double bond
of enone system of chalcones with a thiophene moiety
would enable the molecules to adopt the correct geome-
try for activity. Furthermore, due to the absence of the
a,b-unsaturated ketone moiety, an in vivo Michael reac-
tion should not present an issue.
Replacement of the phenyl ring of compound 2a by the
bioisosteric 2-thienyl group (compound 2b) had minor
effects on antiproliferative activity against the five cell
lines. The isomeric 3-thienyl derivative 2c was less active
than 2b, although the difference between the two com-
pounds was minimal in the Molt-4 cell line.
A series of molecular modelling studies were carried out
on this class of compounds. Structure alignment be-
tween chalcone 1f and thiophene analogue 2m was per-
formed with the Flexible Alignment tool in MOE and
supports our hypothesis that the double bond could be
replaced by the heterocyclic ring as a spacer between
the two aromatic rings (Fig. 1).
Substitution on the phenyl ring had variable effects.
Introduction of a weak electron-withdrawing group
(EWG), a para-fluorine atom (2d), had little overall ef-

R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 5367–5376
5369
OCH3
OCH3
H3CO
OCH3
CHO
Cl
O
OCH3
a
+
R
R
OCH3
R
S
O
3a-p
4a-p
O
2a-p
S -
6
a, R=C6H5
b, R=2-thienyl
c, R=3-thienyl
d, R=p-F-C6H4
e, R=m-F-C6H4
f, R=o-F-C6H4
b
OCH3
g, R=p-Cl-C6H4
OCH3
H3CO
h, R=o-Cl-C6H4
i, R=p-CH3-C6H4
j, R=o-CH3-C6H4
k, R=p-OCH3-C6H4
l, R=m-OCH3-C6H4
m, R=o-OCH3-C6H4
n, R=m,p-2(OCH3)-C6H3
o, R=p-NO2-C6H4
p, R=m-NO2-C6H4
O
S
O
S
5
Scheme 1. Reagents: (a) POCl3, DMF, 65 ꢁC, 5 h; (b) piperidine, EtOH, reflux, 2 h.
Table 1. In vitro inhibitory effects of compounds 1a–g and 2a–p against the proliferation of murine leukaemia (L1210), murine mammary carcinoma
(FM3A), human T-lymphocyte (Molt/4 and CEM) and human cervix carcinoma (HeLa) cells
Compound
IC50 (lM)a
L1210
FM3A/0
Molt4/C8
CEM/0
HeLa
2a
2b
2c
2d
2e
2f
0.56 0.05
0.53 0.11
1.3 0.81
0.59 0.06
2.4 0.21
0.52 0.03
2.2 0.41
0.75 0.12
0.49 0.01
2.2 0.30
2.3 0.20
2.5 0.10
0.51 0.03
0.52 0.01
>10
0.46 0.18
0.46 0.01
1.8 0.30
1.1 0.03
5.6 4.0
0.87 0.46
4.6 0.21
1.7 0.31
0.47 0.04
2.1 0.01
2.4 0.40
2.4 0.42
0.44 0.04
0.96 0.10
>10
0.40 0.02
0.30 0.07
0.37 0.09
0.40 0.09
1.2 0.11
0.33 0.04
1.3 0.06
1.0 0.90
0.38 0.04
0.60 0.05
0.46 0.36
1.8 0.51
0.18 0.09
0.38 0.01
>10
0.48 0.06
0.43 0.08
1.3 1.02
0.56 0.27
2.1 0.20
0.48 0.05
2.3 0.51
0.49 0.07
0.36 0.01
1.2 0.51
2.2 0.51
2.0 0.01
0.37 0.03
0.54 0.10
>10
0.31 0.07
0.47 0.02
1.1 0.31
0.30 0.01
1.7 0.01
1.4 0.01
1.5 0.21
1.6 0.10
0.41 0.03
1.6 0.02
1.0 0.60
1.4 0.11
0.16 0.09
0.50 0.01
>10
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
1a
1b
1c
1d
1e
1f
2.1 0.12
>10
>10
2.4 0.11
>10
>10
1.7 0.11
>10
>10
1.8 0.21
>10
>10
1.4 0.12
>10
>10
>10
>10
>10
>10
>10
2.8 0.42
4.3 1.9
2.1 0.01
8.1 2.3
8.4 1.0
11 1.0
1.4 0.31
3.1 0.11
1.9 0.32
3.1 1.8
1.5 0.11
2.2 0.42
1.8 0.21
1.9 0.21
1.3 0.40
3.7 0.72
1.4 0.22
7.9 0.01
10 2.2
1g
2
0.22
aIC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as means
curves of at least three independent experiments.
SE from the dose–response
fect on antiproliferative activity against L1210, Molt4,
CEM and HeLa cells, but increasing the size of the hal-
ogen from fluorine to chlorine (2g) caused loss of anti-
proliferative activity. Moving the fluorine from the
para (2d) to the ortho position (2f) slightly enhanced
antiproliferative activity in four of the five cell lines,
the exception being the HeLa cells. In contrast, the
meta-fluoro derivative 2e was substantially less potent
than 2d and 2f. The replacement of the para-fluoro
group with a nitro moiety, to give 2o, eliminated anti-
proliferative activity (IC50’s > 10 lM). Shifting the ni-
tro group to the meta position (compound 2p)
enhanced activity with respect to 2o. Compound 2p
had antiproliferative activities comparable to those of
the meta-fluoro derivative 2e against L1210, Molt-4,
CEM and HeLa cell lines. Thus, selected, single EWG’s
can be placed at all three positions in the phenyl ring
with only minor effects on antiproliferative activity,

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R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 5367–5376
but no modification improved activity relative to the
unsubstituted 2a.
compound.16 With the exception of compounds 2f
and 2n, all new compounds were comparable or supe-
rior to CA-4 as inhibitors of tubulin assembly, with
the order of activity being 2b = 2i>2d>2m>CA-4>
2a>2j>2f>2n. Compounds 2b and 2i were the most
active (IC50, 0.8 lM), having twice the potency of
CA-4 (IC50, 1.4 lM), while the others had IC50’s rang-
ing from 1 to 3 lM.
Turning to the effects of an electron-releasing group
(ERG) on the phenyl moiety, we found that a p-methyl
(2i) group caused only minor changes in antiprolifera-
tive activity relative to the unsubstituted 2a. Reduced
activity occurred when the methyl substituent was
moved from the para to ortho position (compound 2j).
In the colchicine binding studies, compounds 2abdfim
potently inhibited the binding of [3H]colchicine to tubu-
lin, since 66–82% inhibition occurred with these agents
at 1 lM and the colchicine at 5 lM. None, however,
was quite as potent as CA-4, which in these experiments
inhibited colchicine binding by 87%. While this group of
compounds were all potent in the biological assays (inhi-
bition of cell growth, tubulin assembly and colchicine
binding), correlations between the three assay types were
imperfect. Thus, while compounds 2b and 2i were the
best inhibitors of tubulin assembly, their effects on col-
chicine binding were matched by compounds 2a and
2m, which were about half as active as assembly
inhibitors.
The antiproliferative activity of the thiophene deriva-
tives can be further characterized in terms of the substi-
tution pattern and the number of methoxy groups on the
phenyl ring (compounds 2k–n ). With the exception of
the Molt-4 cells, p-methoxy derivative (2k) was 3–5-fold
less active than 2a. Moreover, changing the methoxy
group from the para to the meta position (2l) maintained
antiproliferative activity, while potency was consider-
ably increased with the ortho -methoxy derivative (2m),
which was about 3–10-fold more active than 2k and 2l.
With L1210 cells, potency was little changed comparing
2m with the m,p-dimethoxy derivative (2n), but about a
2-fold loss of activity occurred with the other four cell
lines. Moreover, 2n had a greater potency than the
mono-substituted para (2k) and meta (2l) methoxy
derivatives.
The effects on the cell cycle of a selected group of com-
pounds (2abdfijmn) were examined by flow cytometry
after staining of the cells with propidium iodide. K562
(human leukaemia) cells were exposed for 24 h in the
presence of each compound used at a cytostatic concen-
tration (1.3 lM 2a, 1.7 lM 2 b, 2.2 lM 2d, 1.0 lM 2f,
1.4 lM 2i, 4.0 lM 2j, 1.5 lM 2m and 2.1 lM 2n). Table
3 lists the percentage of cells arrested in the G2-M
phase. All compounds caused an increase in the G2-M
peak. As shown in Figure 2, compounds 2a and 2d in-
duced a modest increase of cells in the S and G2-M
phases of the cell cycle relative to the control. Com-
pounds 2bfijmn were the most active in inducing a G2-
M block with a marked decrease of cells in S and G0–
G1. These data suggest that this class of molecules act
selectively on the G2-M phase of the cell cycle. More-
over, derivatives 2j and 2m induced apoptosis to the
greatest extent, as shown by the appearance of a rela-
tively large sub-Go-Gi peak.
By comparing the effects of ERG’s and EWG’s on the
phenyl at the C5-thiophene position, no clear influence
on antiproliferative activity was observed. In fact, sev-
eral compounds characterized by the presence of substit-
uents with opposite electronic effects showed the same
potency. For example, compound 2d containing the
electron-withdrawing fluoro group showed the same
antiproliferative potency as compound 2i containing
the electron-donating methyl group.
The more active compounds (2abdfijmn) 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 latter assay, the com-
pounds and tubulin were examined at a concentration
of 1 lM with the colchicine at 5 lM) (Table 2).14,15
For comparison, the antitubulin agent CA-4 was exam-
ined in contemporaneous experiments as a reference
Molecular docking studies of 2m in the colchicine bind-
ing site of tubulin were performed,12 and the results
showed good overlap of the trimethoxyphenyl moiety
of the thiophene analogue with the corresponding group
Table 2. Inhibition of tubulin polymerization and colchicine binding
by compounds 2abdfijmn and CA-4
Table 3. Percentage of K562 cells in G2-M phase of cell cycle after
24 h treatment with compounds 2abdfijmn used at a cytostatic
concentration
Compound
Tubulin assemblya
Colchicine
bindingb
IC50 SD (lM)
%
SD
2a
2b
1.5 0.1
0.8 0.1
1.1 0.1
2.0 0.1
0.8 0.1
1.7 0.3
1.3 0.1
3.0 0.2
1.4 0.1
74
82
66
3
2
2
Compound
Cytostatic concentration
(lM)
Percentage cells in
G2-M phase (%)
2d
2f
Control
2a
2b
0.0
1.3
1.7
2.2
1.0
1.4
4.0
1.5
2.1
13
18
18
75
21
81
85
62
57
66 0.7
74 0.5
2i
2j
49 5
74 0.5
2d
2f
2m
2n
28
87
4
3
2i
CA-4
2j
a Inhibition of tubulin polymerization. Tubulin was at 10 lM.
2m
2n
b Inhibition of [3H]colchicine binding. Tubulin, colchicine and tested
compound were at 1, 5 and 1 lM, respectively.

R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 5367–5376
5371
trimethoxybenzoyl moieties of chalcones with general
structure 1, enhanced activity of this class of compounds
as antiproliferative agents. All the synthesized com-
pounds inhibited the growth of cancer cell lines at nano-
molar to low micromolar concentrations. Compound
2m, with a ortho-methoxy phenyl group at the 5-position
of thiophene ring, exhibited the greatest antiproliferative
activity among the compounds tested in this study. The
best results for the inhibition of antiproliferative activity
were obtained with the p-F-phenyl (2d), the o-F-phenyl
(2f), the p-CH3-phenyl (2i), the o-OCH3-phenyl (2m)
and the p, m-(OCH3)2-phenyl (2n) derivatives at the 5-
position of the thiophene ring. There was also no clear
difference in effect on activity between an EWG versus
an ERG, once substituent size was taken into account.
We identified tubulin as the molecular target of the mol-
ecules, since the compounds with the greatest inhibitory
effects on cell growth strongly inhibited tubulin assem-
bly and binding of colchicine to tubulin. These activities
differed little from those of CA-4, and all the most active
compounds had quantitatively similar effects in the
tubulin assays, varying within a narrow range (IC50’s
for assembly, 0.8–2 lM with 10 lM tubulin; 49–82%
inhibition of the binding of 5 lM colchicine, with the
inhibitor and tubulin both at 1 lM). We also showed
by flow cytometry that a set of the active compounds
had cellular effects typical of agents that bind to tubulin,
causing the accumulation of cells in the G2/M phase of
the cell cycle. Molecular modelling studies were also per-
formed, and the proposed binding mode for compound
2m in the colchicine site of tubulin was consistent with
the experimental data.
5. Experimental
5.1. Chemistry
Figure 2. Effects of compounds 2a (panel b), 2b (c), 2d (d), 2f (e), 2i (f),
2j (g), 2m (h) and 2n (i), on DNA content/cell following treatment of
K562 cells for 24 h. The cells were cultured without compound
(control, panel a) or with each compound used at a cytostatic
concentration. Cell cycle distribution was analyzed by the standard
propidium iodide procedure. Control cells at the sub-G0–G1 (labelled
A), G0–G1, S, and G2-M phases of the cell cycle are indicated in panel
(a).
5.1.1. Materials and methods. All acetophenones are
commercially available and were used as received. H
1
and 13C NMR spectra were recorded on a Bruker AC
200 and Varian 400 Mercury Plus spectrometer, respec-
tively. Chemical shifts (d) are given in ppm upfield from
tetramethylsilane as internal standard, and the spectra
were recorded in appropriate deuterated solvents, as
indicated. Positive-ion electrospray ionization (ESI)
mass spectra were recorded on a double-focusing Finn-
igan MAT 95 instrumental with BE geometry. Melting
points (mp) were determined on a Buchi–Tottoli appa-
ratus and are uncorrected. All products reported showed
1H NMR spectra in agreement with the assigned struc-
tures. Elemental analyses were conducted by the Micro-
analytical Laboratory of the Chemistry Department of
the University of Ferrara. All reactions were carried
out under an inert atmosphere of dry nitrogen, unless
otherwise described. 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 chromatogra-
phy was performed using 230–400 mesh silica gel and
the indicated solvent system. Organic solutions were
dried over anhydrous Na2SO4.
of the DAMA–colchicine co-crystallized with the tubu-
lin. A hydrogen bond was also formed between the
ortho-methoxy group of 2m and bAsn101 (residue num-
bering as in the crystal structure used), suggesting a pos-
sible structural explanation for the reduced activity of
2k and 2l where the methoxy group was present in the
para- and meta-positions, respectively, on the aromatic
ring (Fig. 3).
4. Conclusions
In conclusion, we have discovered a new class of simple
synthetic inhibitors of tubulin polymerization, based on
a
2-(30,40,50-trimethoxybenzoyl)-5-aryl/heteroaryl-thio-
phene molecular skeleton. SAR studies revealed that
the replacement of the double bond of the enone system
with a thiophene nucleus between the aryl and 30,40,50-

5372
R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 5367–5376
Figure 3. Proposed binding mode of 2m. DAMA–colchicine is represented in green.
5.1.2. General procedure A for the synthesis of compounds
4a–p. POCl3 (3.1 g, 20 mmol) was added dropwise over
20 min, maintaining the temperature below 25 ꢁC, to an
ice-cooled stirred solution of dry DMF (10 mL). After
10 min, the appropriate acetophenone derivative (5 mmol)
dissolved in DMF (5 mL) was added dropwise to the
DMF-POCl3 complex. The reaction mixture was stirred
for 1 h at 5 ꢁC, heated at 70 ꢁC for 5 h, cooled at room
temperature and finally poured in a ice-cold saturated
NaOAc water solution (20 mL). The resulting mixture
was extracted with ethyl acetate (3 · 15 mL). The organic
phase was washed with water (3 · 10 mL), brine (10 mL),
dried over Na2SO4 and concentrated in vacuo. The result-
ing residue was purified by silica gel column chromatogra-
phy to yield b-chlorocinnamaldehydes 4a–p.
For the characterization (1H NMR) of (Z)-3-chloro-3-
(3,4-dimethoxyphenyl)acrylaldehyde (4n): Dressler, V.;
Bodendorf, K. Arch. Pharm. 1970, 6, 481.
For the characterization (1H NMR) of (Z)-3-chloro-3-
(4-nitrophenyl)acrylaldehyde (4o): Rappoport, Z.; Gaz-
it, A. J. Org. Chem. 1986, 51, 4112.
5.1.3. (Z)-3-Chloro-3-(thiophen-2-yl)acrylaldehyde (4b).
Following general procedure A, compound 4b was
purified by chromatography eluting with petroleum
ether–ethyl acetate (9:1). Yellow solid, yield: 42%, mp
1
38–40 ꢁC. H NMR (CDCl3) d: 6.59 (d, J = 6.8 Hz,
1H), 7.13 (dd, J = 4.8 and 3.6 Hz, 1H), 7.55 (dd, J = 4.8
and 1.2 Hz, 1H), 7.67 (dd, J = 4.8 and 1.2 Hz, 1H), 10.1
(d, J = 6.8 Hz, 1H).
For the characterization (1H NMR) of (Z)-3-chloro-3-
phenylacrylaldehyde (4a) see: Ali, M. M.; Sana, S.; Tas-
neem; R. K. C.; Saiprakash, P. K. Synth. Commun.
2002, 32, 1351.
5.1.4. (Z)-3-Chloro-3-(thiophen-3-yl)acrylaldehyde (4c).
Following general procedure A, compound 4c was puri-
fied by chromatography eluting with petroleum ether–
ethyl acetate (9:1). Yellow solid, yield: 68%, mp 77–79
For the characterization (1H NMR) of (Z)-3-chloro-3-p-
tolylacrylaldehyde (4i) see: Miura, M.; Okuro, K.; Hat-
tori, A.; Nomura, M. J. Chem. Soc., Perkin Trans. 1
1989, 73.
1
ꢁC. H NMR (CDCl3) d: 6.60 (d, J = 6.8 Hz, 1H), 7.34
(dd, J = 5.0 and 1.4 Hz, 1H), 7.55 (dd, J = 5.2 and
3.0 Hz, 1H), 7.94 (dd, J = 3.0 and 1.6 Hz, 1H), 10.1 (d,
J = 6.8 Hz, 1H).
For the characterization (1H NMR) of (Z)-3-chloro-3-o-
tolylacrylaldehyde (4j): Beaton, C. M.; Chapman, N. B.;
Clarke, K.; Willis, J. M. J. Chem. Soc., Perkin Trans. 1
1976, 2355.
5.1.5. (Z)-3-Chloro-3-(4-fluorophenyl)acrylaldehyde (4d).
Following general procedure A, compound 4d was puri-
fied by chromatography eluting with petroleum ether–
ethyl acetate (8:2). Brown oil, yield: 52%. 1H NMR
(CDCl3) d: 6.60 (d, J = 7.0 Hz, 1H), 7.15 (t, J = 8.2
Hz, 2H), 7.76 (dd, J = 8.6 and 5.2 Hz, 2H), 10.1 (d,
J = 7.0 Hz, 1H).
For the characterization (1H NMR) of (Z)-3-chloro-3-
(3-methoxyphenyl)acrylaldehyde (4l): Fringuelli, F.;
Serena, B.; Taticchi, A. J. Chem. Soc., Perkin Trans. 2
1980, 971.
5.1.6. (Z)-3-Chloro-3-(3-fluorophenyl)acrylaldehyde (4e).
Following general procedure A, compound 4e was puri-
fied by chromatography eluting with petroleum ether–
ethyl acetate (9:1). Yellow solid, yield: 60%, mp 40–42
For the characterization (1H NMR) of (Z)-3-chloro-3-
(2-methoxyphenyl)acrylaldehyde (4m): Chakraborty,
A; Ray, J. K. Synth. Commun. 1995, 25, 1869.

R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 5367–5376
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1
ꢁC. H NMR (CDCl3) d: 6.65 (d, J = 6.6 Hz, 1H), 7.18
(m, 1H), 7.43 (m, 3H), 10.1 (d, J = 6.6 Hz, 1H).
5.2.1. (3,4,5-Trimethoxyphenyl)-(5-phenylthiophen-2-yl)-
methanone (2a). Following procedure B, compound 2a
was isolated as a yellow oil. Yield: 57%. 1H NMR
(CDCl3) d: 3.93 (s, 3H), 3.95 (s, 6H), 7.16 (s, 2H), 7.37
(d, J = 4.0 Hz, 1H), 7.43 (m, 3H), 7.67 (d, J = 4.0 Hz,
1H), 7.69 (m, 2H). 13C NMR (100 MHz, CDCl3) d:
56.4 (2·), 61.1, 106.8 (2·), 123.9 (2·), 126.4 (2·), 129.2
(2·), 133.4 (2·), 135.6 (2·), 141.9 (2·), 153.1 (2·),
187.0. MS-ESI (m/z): 355.3 [M+1]+. Anal. Calcd for
C20H18O4S: C, 67.78; H, 5.12. Found: C, 67.56; H, 5.01.
5.1.7. (Z)-3-Chloro-3-(2-fluorophenyl)acrylaldehyde (4f).
Following general procedure A, compound 4f was puri-
fied by chromatography eluting with petroleum ether–
ethyl acetate (8:2). Brown oil, yield: 51%. 1H NMR
(CDCl3) d: 6.68 (d, J = 6.8 Hz, 1H), 7.17 (m, 2H),
7.42 (m, 1H), 7.73 (t, J = 7.8 Hz, 1H), 10.3 (d, J = 6.8
Hz, 1H).
5.1.8. (Z)-3-Chloro-3-(4-chlorophenyl)acrylaldehyde (4g).
Following general procedure A, compound 4g was puri-
fied by chromatography eluting with petroleum ether–
ethyl acetate (9:1). Yellow solid, yield: 58%, mp 90–92
ꢁC. H NMR (CDCl3) d: 6.65 (d, J = 6.8 Hz, 1H), 7.43
(dd, J = 6.8 and 1.8 Hz, 1H), 7.72 (d, J = 6.8 and 1.8,
2H), 10.2 (d, J = 6.8 Hz, 1H).
5.2.2. (3,4,5-Trimethoxyphenyl)(5-(thiophen-2-yl)thioph-
en-2-yl)methanone (2b). Following general procedure B,
compound 2b was isolated as a yellow solid, yield: 38%;
mp 94–96 ꢁC. 1H NMR (CDCl3) d: 3.92 (s, 3H), 3.94 (s,
6H), 7.02 (m, 1H), 7.13 (s, 2H), 7.23 (d, J = 4.0 Hz, 1H),
7.35 (m, 2H), 7.60 (d, J = 4.0 Hz, 1H). 13C NMR
(100 MHz, CDCl3) d: 56.4 (2·), 61.1, 106.7 (2·), 124.2
(2·), 125.9, 126.7 (2·), 128.4 (2·), 133.2, 135.5, 136.3,
146.2, 153.1, 186.8. MS-ESI (m/z): 361.4 [M+1]+. Anal.
Calcd for C18H16O4S2: C, 59.98; H, 4.47. Found: C,
59.78; H, 4.35.
1
5.1.9. (Z)-3-Chloro-3-(3-chlorophenyl)acrylaldehyde (4h).
Following general procedure A, compound 4h was puri-
fied by chromatography eluting with petroleum ether–
ethyl acetate (9:1). Yellow oil, yield: 68%. 1H NMR
(CDCl3) d: 6.38 (d, J = 7.0 Hz, 1H), 7.44 (m, 4H),
10.2 (d, J = 7.0 Hz, 1H).
5.2.3. (3,4,5-Trimethoxyphenyl)(5-(thiophen-3-yl)thioph-
en-2-yl)methanone (2c). Following general procedure B,
compound 2c was isolated as a pink solid. Yield: 54%;
mp 122–124 ꢁC. 1H NMR (CDCl3) d: 3.92 (s, 3H),
3.94 (s, 6H), 7.12 (s, 2H), 7.24 (d, J = 3.8 Hz, 1H),
7.38 (m, 2H), 7.59 (m, 1H), 7.62 (d, J = 3.8 Hz,
1H).13C NMR (100 MHz, CDCl3) d: 56.4 (2·), 61.1,
106.7 (2·), 122.3, 123.9 (2·), 126.0, 127.2, 133.3, 134.8,
135.6 (2·), 141.3, 147.8, 153.1, 187.0. MS-ESI (m/z):
361.3 [M+1]+. Anal. Calcd for C18H16O4S2: C, 59.98;
H, 4.47. Found: C, 59.68; H, 4.32.
5.1.10. (Z)-3-Chloro-3-(4-methoxyphenyl)acrylaldehyde
(4k). Following general procedure A, compound 4k was
purified by chromatography eluting with petroleum
ether–ethyl acetate (9:1). Yellow solid, yield: 54%, mp
1
58–60 ꢁC. H NMR (CDCl3) d: 3.87 (s, 3H), 6.61 (d,
J = 6.8 Hz, 1H), 6.94 (dd, J = 6.8 and 2.0 Hz, 1H),
7.72 (d, J = 6.8 and 2.0, 2H), 10.2 (d, J = 6.8 Hz, 1H).
5.1.11. (Z)-3-Chloro-3-(3-nitrophenyl)acrylaldehyde (4p).
Following general procedure A, compound 4p was puri-
fied by chromatography eluting with petroleum ether–
ethyl acetate (2:8). Yellow solid, yield: 80%, mp 81–83
ꢁC. H NMR (CDCl3) d: 6.76 (d, J = 6.8 Hz, 1H), 7.73
(t, J = 8.0 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 8.38 (dd,
J = 8.0 and 1.4 Hz, 1H), 8.64 (d, J = .4 Hz, 1H), 10.2 (d,
J = 6.8 Hz, 1H).
5.2.4. (5-(4-Fluorophenyl)thiophen-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (2d). Following general procedure B,
compound 2d was isolated as a white solid. Yield: 48%,
mp 127–128 ꢁC. 1H NMR (CDCl3) d: 3.93 (s, 6H),
3.95 (s, 3H), 7.15 (s, 2H), 7.18 (t, J = 7.8 Hz, 2H), 7.29
(d, J = 3.8 Hz, 1H), 7.63 (d, J = 3.8 Hz, 1H), 7.68 (m,
2H). 13C NMR (100 MHz, CDCl3) d: 56.4 (2·), 61.1,
106.8 (2·), 116.2, 116.4, 123.9, 128.2, 128.3, 129.7,
133.2, 135.6 (2·), 141.9, 142.2, 151.9, 153.1 (2·), 187.0.
MS-ESI (m/z): 373.5 [M+1]+. Anal. Calcd for
C20H17FO4S: C, 64.50; H, 4.60; F, 5.10. Found: C,
64.33; H, 4.51; F, 4.89.
1
5.2. General procedure B for the synthesis of 5-aryl/
heteroaryl-thiophen-2-yl-(3,4,5-trimethoxyphenyl)-metha-
nones 2a–p
Piperidine (0.2 mL, 2 mmol) was added to a stirred solu-
tion of dithiocarbonic acid O-ethyl ester S-[2-oxo-2-
(3,4,5-trimethoxyphenyl)-ethyl] ester 5 (330 mg, 1 mmol)
dissolved in ethanol (20 mL). The reaction mixture was
stirred for 30 min at room temperature. The correspond-
ing b-chlorocinnamaldehyde (1 mmol) was added, and
the solution was stirred at reflux for 3 h, after which
ethanol was removed under reduced pressure and the
residue dissolved in dichloromethane (15 mL). The or-
ganic phase was washed with a 5% v/v solution of HCl
(2 · 5 mL), a saturated solution of sodium carbonate
(2 · 5 mL), brine (5 mL) and dried over sodium sulfate.
After concentration under reduced pressure, the residue
was purified by silica gel column chromatography
(petroleum ether–EtOAc, 7:3) to furnish the correspond-
ing final compounds 2a–p.
5.2.5. (5-(3-Fluorophenyl)thiophen-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (2e). Following general procedure B,
compound 2e was isolated as a white solid. Yield: 46%,
mp 129–131 ꢁC. 1H NMR (CDCl3) d: 3.93 (s, 6H),
3.95 (s, 3H), 7.16 (s, 2H), 7.36 (d, J = 4.0 Hz, 1H),
7.46 (m, 4H), 7.68 (d, J = 4.0 Hz, 1H). 13C NMR
(100 MHz, CDCl3) d: 56.4 (2·), 61.0, 106.8 (2·), 113.1,
113.4, 115.9, 116.1, 122.2, 124.5, 130.8, 130.9, 133.1,
135.4, 141.9, 142.8, 151.3, 153.1, 186.9. MS-ESI (m/z):
373.4 [M+1]+. Anal. Calcd for C20H17FO4S: C, 64.50;
H, 4.60; F, 5.10. Found: C, 64.41; H, 4.48; F, 4.93.
5.2.6. (5-(2-Fluorophenyl)thiophen-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (2f). Following general procedure B,

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R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 5367–5376
compound 2e was isolated as a white solid. Yield: 48%,
mp 123–125 ꢁC. 1H NMR (CDCl3) d: 3.93 (s, 3H),
3.94 (s, 6H), 7.10 (m, 1H), 7.18 (s, 2H), 7.32 (m, 1H),
7.42 (d, J = 3.8 Hz, 1H), 7.56 (m, 1H), 7.64 (m, 1H),
7.68 (d, J = 3.8 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d: 56.4 (2·), 61.1, 106.8 (2·), 116.6, 116.8, 124.8, 127.1,
127.2, 129.1, 130.4, 130.5, 133.2, 135.0, 141.9, 142.6,
145.8, 153.1, 187.1. MS-ESI (m/z): 373.5 [M+1]+. Anal.
Calcd for C20H17FO4S: C, 64.50; H, 4.60; F, 5.10.
Found: C, 64.41; H, 4.48; F, 4.90.
5.2.11. (3,4,5-Trimethoxyphenyl)(5-(4-methoxyphenyl)-
thiophen-2-yl)methanone (2k). Following general proce-
dure B, compound 2k was isolated as a white solid. Yield:
34%; mp 93–96 ꢁC. 1H NMR (CDCl3) d: 3.86 (s, 3H), 3.93
(s, 6H), 3.94 (s, 3H), 6.91 (d, J = 8.8 Hz, 2H), 7.14 (s, 2H),
7.22 (d, J = 4.0 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.66 (d,
J = 4.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) d:55.5,
56.4 (2·), 61.0, 105.8, 106.7 (2·), 114.6 (2·), 122.8,
126.1, 127.8 (2·), 132.5, 133.4, 135.9, 141.1, 153.1,
153.4, 160.6, 187.1. MS-ESI (m/z): 385.3 [M+1]+. Anal.
Calcd for C21H20O5S: C, 65.61; H, 5.24. Found: C,
65.44; H, 5.03.
5.2.7. (5-(4-Chlorophenyl)thiophen-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (2g). Following general procedure B,
compound 2g was isolated as a white solid. Yield: 62%,
mp 137–139 ꢁC. 1H NMR (CDCl3) d: 3.93 (s, 3H),
3.96 (s, 6H), 7.15 (s, 2H), 7.34 (d, 4.0 Hz, 1H), 7.43 (d,
J = 8.6 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.66 (d,
4.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) d : 56.4
(2·), 61.1, 106.8 (2·), 124.2, 127.6 (2·), 129.5 (2·),
131.9, 133.1, 135.1, 135.5, 141.9, 142.5, 151.6, 153.1
(2·), 186.9. MS-ESI (m/z): 389.9 [M+1]+. Anal. Calcd
for C20H17ClO4S: C, 61.77; H, 4.41; Cl, 9.12. Found:
C, 61.63; H, 4.33; Cl, 8.96.
5.2.12.(3,4,5-Trimethoxyphenyl)(5-(3-methoxyphenyl)thio-
phen-2-yl)methanone (2l). Following general procedure B,
compound 2l was isolated as a white solid, yield: 53%; mp
138–140 ꢁC. 1H NMR (CDCl3) d: 3.87(s, 3H), 3.92(s, 3H),
3.94 (s, 6H), 6.92 (s, 1H), 7.16 (s, 2H), 7.21 (s, 1H), 7.30 (m,
2H), 7.37(d, J = 3.8 Hz, 1H), 7.65 (d, J = 3.8 Hz, 1H). 13C
NMR (100 MHz, CDCl3) d: 55.5, 56.4 (2·), 61.1, 106.8
(2·), 112.0 (2·), 114.7 (2·), 118.9, 124.1, 130.3, 133.2,
134.6, 135.5, 142.1, 153.1 (2·), 160.1, 187.0. MS-ESI (m/
z): 385.4 [M+1]+. Anal. Calcd for C21H20O5S: C, 65.61;
H, 5.24. Found: C, 65.50; H, 5.11.
5.2.8. (5-(2-Chlorophenyl)thiophen-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (2h). Following general procedure B,
compound 2h was isolated as a yellow solid, yield:
37%; mp 210–212 ꢁC. 1H NMR (CDCl3) d: 3.94 (s,
3H), 3.95 (s, 6H), 7.12 (m, 1H), 7.18 (s, 2H), 7.31
(m, 1H), 7.42 (d, J = 3.8 Hz, 1H), 7.56 (m, 1H),
7.64 (d, J = 9.0 Hz, 1H), 7.69 (d, J = 3.8 Hz, 1H).
13C NMR (100 MHz, CDCl3) d: 56.2, 56.4, 61.1,
106.9 (2·), 126.5, 127.3, 128.6, 129.3, 129.7, 130.0,
130.3, 130.9, 131.4, 132.0, 133.2, 134.4, 152.5, 153.1,
187.1. MS-ESI (m/z): 389.3 [M+1]+. Anal. Calcd for
C20H17ClO4S: C, 61.77; H, 4.41; Cl, 9.12. Found: C,
61.71; H, 4.193; Cl, 9.01.
5.2.13.(3,4,5-Trimethoxyphenyl)(5-(2-methoxyphenyl)thio-
phen-2-yl)methanone (2m). Following general procedure
B, compound 2m was isolated as a brown solid, yield:
69%; mp 135–137 ꢁC. 1H NMR (CDCl3) d: 3.93 (s,
3H), 3.94 (s, 3H), 3.98 (s, 6H), 7.05 (t, J = 7.6 Hz, 1H),
7.18 (s, 2H), 7.22 (d, J = 8.4 Hz, 1H), 7.37 (t,
J = 7.6 Hz, 1H), 7.54 (d, J = 4.2 Hz, 1H), 7.66 (d,
J = 4.2 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H). 13C NMR
(100 MHz, CDCl3) d: 55.7, 56.4 (2·), 61.1, 106.8 (2·),
111.8, 121.1 (2·), 125.8, 128.7, 130.2, 130.5, 133.7,
134.5, 141.6, 142.0, 148.5, 153.0, 156.2, 187.6. MS-ESI
(m/z): 385.6 [M+1]+. Anal. Calcd for C21H20O5S: C,
65.61; H, 5.24. Found: C, 65.44; H, 5.12.
5.2.9 . (3,4,5-Trimethoxyphenyl)(5-p-tolylthiophen-2-yl)-
methanone (2i) . Following general procedure B, com-
pound 2i was isolated as a white solid. Yield: 47%; mp
135–137 ꢁC. H NMR (CDCl3) d: 2.40 (s, 3H), 3.93 (s,
3H), 3.94 (s, 6H), 7.15 (s, 2H), 7.22 (d, J = 8.4 Hz,
2H), 7.32 (d, J = 4.0 Hz, 1H), 7.57 (d, J = 8.4 Hz,
2H), 7.65 (d, J = 4.0 Hz, 1H). 13C NMR (100 MHz,
CDCl3) d: 21.4, 56.4 (2·), 61.1, 106.8 (2·), 123.4 (2·),
126.3 (2·), 129.9 (2·), 130.6, 133.4, 135.7 (2·), 139.5,
141.6, 153.1, 153.5, 187.0. MS-ESI (m/z): 369.4 [M+1]+.
Anal. Calcd for C21H20O4S: C, 68.46; H, 5.47. Found:
C, 68.34; H, 5.36.
5.2.14. (3,4,5-Trimethoxyphenyl)(5-(3,4-dimethoxyphe-
nyl)thiophen-2-yl)methanone (2n). Following general pro-
cedure B, compound 2n was isolated as a yellow solid,
yield: 53%; mp 133–135 ꢁC. H NMR (CDCl3) d: 3.92
(s, 6H), 3.94 (s, 3H), 3.96 (s, 6H), 6.92 (d, J = 8.4 Hz,
1H), 7.15 (s, 2H), 7.17 (s, 1H), 7.28 (d, J = 3.8 Hz, 1H),
7.30 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 3.8 Hz, 1H). 13C
NMR (100 MHz, CDCl3) d: 45.6, 56.1, 56.4 (2·), 61.1,
106.3, 106.8, 109.5, 110.3, 111.6, 119.3, 123.1, 126.4,
130.6, 133.4, 135.8, 141.4, 141.8, 149.4, 153.1, 153.2,
187.0. MS-ESI (m/z): 415.4 [M+1]+. Anal. Calcd for (C
22H22O6S): C, 63.75; H, 5.35. Found: C, 63.61; H, 5.17.
1
1
5.2.10. (3,4,5-Trimethoxyphenyl)(5-o-tolylthiophen-2-yl)-
methanone (2j). Following general procedure B, com-
pound 2j was isolated as a white solid. Yield: 38%; mp
5.2.15. (3,4,5-Trimethoxyphenyl)(5-(4-nitrophenyl)thio-
phen-2-yl)methanone (2o). Following general procedure
B, compound 2o was isolated as a yellow solid. Yield:
25%, mp 162–165ꢁC. 1H NMR (CDCl3) d: 3.87 (s,
3H), 3.94 (s, 6H), 7.16 (s, 2H), 7.52 (d, J = 4.0 Hz,
1H), 7.76 (d, J = 8.8 Hz, 2H), 8.28 (d, J = 8.8 Hz,
2H), 8.34 (d, J = 4.0 Hz, 1H). 13C NMR (100 MHz,
CDCl3) d: 56.5 (2·), 61.1, 106.9 (2·), 124.7 (2·), 126.0,
126.9 (2·), 132.7, 135.3, 139.4, 142.3, 144.6, 147.8,
149.2, 153.2 (2·), 186.8. MS-ESI (m/z): 400.5 [M+1]+.
1
99–101 ꢁC. H NMR (CDCl3) d: 2.48 (s, 3H), 3.94 (s,
3H), 3.95 (s, 6H), 7.09 (m, 1H), 7.13 (d, J = 4.0 Hz,
1H), 7.18 (s, 2H), 7.32 (m, 2H), 7.44 (m, 1H), 7.70 (d,
J = 4.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) d:
21.3, 56.4 (2·), 61.1, 106.8 (2·), 125.6, 126.3, 127.5,
128.0, 129.0, 130.1, 130.2, 130.3, 131.2, 133.2, 134.8,
141.8, 152.5, 153.1, 187.1. MS-ESI (m/z): 369.3 [M+1]+.
Anal. Calcd for C21H20O4S: C, 68.46; H, 5.47. Found:
C, 68.28; H, 5.31.

R. Romagnoli et al. / Bioorg. Med. Chem. 16 (2008) 5367–5376
5375
Anal. Calcd for C20H17NO6S: C, 60.14; H, 4.29; N, 3.51.
Found: C, 60.02; H, 4.10; N, 3.38.
blastic leukaemia) by flow cytometric analysis after
staining with propidium iodide. Cells were exposed
24 h to each compound used at a concentration corre-
sponding to the cytostatic concentration. After treat-
ment cells were washed once in ice-cold PBS and
resuspended at 1 · 106 mL in a hypotonic fluorochrome
solution containing propidium iodide (Sigma) 50 lg/mL
in 0.1% sodium citrate plus 0.03% (v/v) nonidet P-40
(Sigma). After 30 min of incubation, the fluorescence
of each sample was analyzed as single-parameter fre-
quency histograms by using a FACScan flow cytometer
(Becton–Dickinson, San Jose, CA). The distribution of
cells in the cell cycle was analyzed with the ModFit
LT3 program (Verity Software House, Inc.).
5.2.16. (3,4,5-Trimethoxyphenyl)(5-(3-nitrophenyl)thio-
phen-2-yl)methanone (2p). Following general procedure
B, compound 2p was isolated as a white solid. Yield:
43%, mp 141–143 ꢁC. 1H NMR (CDCl3) d: 3.94 (s,
6H), 3.96 (s, 3H), 7.17 (s, 2H), 7.48 (d, J = 4.0 Hz,
1H), 7.64 (s, 1H), 7.72 (d, J = 4.0 Hz, 1H), 7.98 (dd,
J = 7.8 and 1.8 Hz, 1H), 8.22 (dd, J = 7.8 and 1.8 Hz,
1H), 8.54 (t, J = 1.8 Hz, 1H). 13C NMR (100 MHz,
CDCl3) d: 56.5 (2·), 61.1, 106.9 (2·), 121.0 (2·), 123.5
(2·), 125.5 (2·), 130.4 (2·), 132.0, 132.8, 135.3, 143.8,
149.4, 153.2, 186.8. MS-ESI (m/z): 400.5 [M+1]+. Anal.
Calcd for C20H17NO6S: C, 60.14; H, 4.29; N, 3.51.
Found: C, 60.00; H, 4.12; N, 3.18.
5.6. Molecular modelling
5.3. Growth inhibitory activity
All molecular modelling studies were performed on a
MacPro dual 2.66 GHz Xeon running Ubuntu 7, using
Molecular Operating Environment (MOE) 2006.08.17
The tubulin structure was downloaded from the PDB
data bank (http://www.rcsb.org/-PDBcode:1SA0).18
Hydrogen atoms were added to the protein with MOE
and minimized keeping all the heavy atoms fixed until
Murine leukaemia L1210, murine mammary carcinoma
FM3A, human T-lymphocyte Molt 4 and CEM and hu-
man cervix carcinoma (HeLa) cells were suspended at
300,000–500,000 cells/mL of culture medium, and
100 lL of a cell suspension was added to 100 lL of an
appropriate dilution of the test compounds in wells of
microtiter plates. After incubation at 37 ꢁC for two days,
cell number was determined using a Coulter counter.
ꢀ1
a RMSD gradient of 0.05 kcal molꢀ1
A
was reached.
˚
Ligand structures were built with MOE and minimized
using the MMFF94x forcefield until a RMSD gradient
ꢀ1
was reached. The docking simu-
of 0.05 kcal molꢀ1
A
˚
To determine the growth inhibitory activity of the tested
compounds on human myeloid leukaemia K562 cell
line, 2 · 105 cells were plated into 25 mm wells (Costar,
Cambridge, UK) in 1 mL of complete medium and trea-
ted with different concentrations of each drug. After
24 h of incubation, the number of viable cells was deter-
mined using a Coulter counter.
lations were performed using the Alpha Triangle place-
ment method and the London dG scoring method. 300
results for each ligand were generated, and the position
of the trimethoxyphenyl moiety was compared with ring
A of the colchicine analogue co-crystallized with the
protein, discarding the results with a RMSD value
19
˚
>3 A. The best scored result of the remaining confor-
mations for each ligand was further analyzed. The pro-
tein/ligand complexes were minimized using the
MMFF94x force field, until a RMSD gradient of
The IC50 was defined as the compound concentration re-
quired to inhibit cell proliferation by 50%.
ꢀ1
was reached.
0.1 kcal molꢀ1
A
˚
5.4. Effects on tubulin polymerization and on colchicine
binding to tubulin
References and notes
Bovine brain tubulin was purified as described previ-
ously.14 To evaluate the effect of the compounds on
tubulin assembly in vitro, varying concentrations were
preincubated with 10 lM tubulin in glutamate buffer at
30 ꢁC and then cooled to 0 ꢁC. After the addition of
GTP, the mixtures were transferred to 0 ꢁC cuvettes in a
recording spectrophotometer and warmed to 30 ꢁC, and
the assembly of tubulin was observed turbidimetrically.
The IC50 was defined as the compound concentration that
inhibited the extent of assembly by 50% after a 20 min
incubation. The capacity of the test compounds to inhibit
colchicine binding to tubulin was measured as described,15
except that the reaction mixtures contained 1 lM tubulin,
5 lM [3H]colchicine and 1 lM test compound.
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5376
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Claisen–Schmidt aldol condensation of 3,4,5-trimethoxy-
acetophenone and the corresponding aldehydes in the
presence of sodium hydroxide. These compounds have
17. Molecular Operating Environment (MOE). Version
2007.09, Chemical Computing Group, Inc., Montreal,
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19. Code ‘fragment_rmsd.svl’ obtained from SLV Exchange
website http://svl.chemcomp.com, Chemical Computing
Group, Inc., Montreal, Canada.