1. dia

1
Synthesis of thiophene and a-terthiophene
derivatives with antiproliferative
activity Zsolt Székelyhidia, János Patób,
László Orfia,b,c, Frigyes Wáczeka, Péter
Bánhegyia, Gyöngyi Bökönyid, Edit Z. Szabód, Edit
Várkondia, Ákos Papa, Richárd Schwába and György
Kéria,b,d aCooperative Research Centre,
Semmelweis University, Rippl-Rónai u. 37., 1062
Budapest, Hungary bVichem Chemie Ltd., Hermann u.
15., 1022 Budapest, Hungary cDept. of
Pharmaceutical Chemistry, Semmelweis University,
Hogyes u. 9., 1092 Budapest, Hungary dPeptide
Biochemistry Research Group, Hung. Acad. Sci -
Semmelweis Univ., Puskin u. 9., 1088 Budapest,
Hungary Tel. [phone] Fax [phone]
szekelyhidi_at_kkk.sote.hu
Introduction The uncontrolled growth of tumour
cells has been linked in many cases to
inappropriate signal transduction. Targeting the
signal transduction process in order to control
tumour growth has become an attractive area of
drug research. Kinases are the most important
signalling enzymes and most of the signal
transduction therapy based drug research is aimed
to inhibit the false or overexpressed signal of
certain pathologically relevant kinases1.
Thousands of compounds around several scaffolds
have been developed as potential kinase
inhibitory drugs. A few terthiophenes are known
as PKC inhibitors2,3 and nitrile derivatives of
thiophene inhibit EGF-RTK (epidermal growth
factor receptor tyrosine kinase)4,5. Both kinases
are involved in cell proliferation, thus we have
planned and synthesised a series of new thiophene
and terthiophene derivatives as well as some of
known structurally related PKC inhibitors (4a,
10, 11, 14), and studied them in cell
proliferation test on EGFR overexpressing tumour
cell line (A431). EGFR (epidermal growth factor
receptor) is a transmembrane protein composed of
three domains the extracellular ligand-binding
domain, a transmembrane region and an
intracellular protein kinase domain. A number of
different ligands, including EGF, neuregulins,
heparinbinding EGF, ß-cellulin and transforming
growth factor-a have shown the capability to bind
to the extracellular domain of EGFR. The binding
of the ligand to EGFR induces the formation of
active dimers that are able to turn on a
transduction signal through the cell. EGFR
activation lies in the multiple processes
involved in cell proliferation, adhesion,
migration, development of angiogenesis, and
inhibition of apoptosis.4
Fig.1
Biological test
Human A431 epidermoid carcinoma cells were
cultured in DMEM (Dulbeccos Mod Eagle Medium)
supplemented with 10 FCS (foetal calf serum),
200 mM L-glutamine, 10000 U/ml penicillin and 10
mg/ml streptomycin (Gibco Life Sci) at 37C and
5 CO2. Cells were seeded into 96-well plates and
incubated for 16 hours before serial dilutions of
compounds were added. Cells were treated for 6
and 48 hours. Cells used for 6 hour and 48 hour
treatment were seeded at 4x104 and 1x104 per well
respectively. Antiproliferative efficacy of the
compounds was analysed with Methylene blue
test8. All compounds were dissolved in DMSO and
diluted in cell culture medium for
the-proliferation tests in final concentrations
of 50, 10, 2, 0.4, 0.08 µM and tested in
duplicates. Cycloheximide, a well established
inducer of apoptosis was used as positive
control. Antiproliferative effect was first
expressed as a percentage of the optical density
(OD) of treated (T) and control (C) wells after
both 6 and 48 hours (T/C100). Because new
protein synthesis is required for apoptosis in
immortalized cell lines, compounds that induce
programmed cell death will show significantly
less antiproliferative activity after 6h than
after 48h. In the optimal case an apoptosis
inducing compound will cause 100 viability after
6h and 0 after 48h. Therefore, analysing T/C48
versus T/C6h will correlate with the apoptosis
inducing specificity of a compound. Cut-off
limit for effective compounds was set for
differences expressed as (T/C6h - T/C48) gt 80.
IC50 values were generated from IC50 graphs.
Reagents and conditions (a) AlCl3, DCM, 0 0C
(b) Lawessons reagent
We have tried the synthetic route described in
the literature to prepare a-terthiophene.
According to this method succinyl chloride 1 was
reacted with thiophene 2 in the presence of
aluminium chloride in dichloromethane at 0 0C to
obtain the dithiophene-1,4-diketone 3 in 80
yield. Thionation using Lawessons reagent
yielded ?-terthiophene 4a in 90.2 Unfortunately,
the yield of the first step was much less than
expected and the product was contaminated.
Moreover, this route was not suitable to obtain
asymmetric terthiophene analogs. For this reason
the following synthetic route was developed.
Thiophene-2-carboxaldehyde 5 paraformaldehyde and
a secondary amines in ethanol produced
Mannichbases 6, 7 and 8. The Mannich-bases were
reacted with thiophene-carboxaldehyde and NaCN in
abs. DMF to obtain dithiophene-diketones 3, 9.
Thionation using phosphorus pentasulfide yielded
terthiophene 4a and 4b.
Fig.2
Reagents and conditions (a) ethanol, secondary
amine, 12 h, reflux temperature (b)
thiophene-2-carboxaldehyde (b)
thiophene-3-carboxaldehyde NaCN, abs. DMF, 12 h,
room temperature (c) P2S5, NaHCO3,
THF/hexane3/1, 12 h, room temperature
?-Terthiophene 4a was lithiated with LDA in abs.
THF under argon at 78 degree then the lithium
compound was quenched with DMF to give
?-terthiophenedialdehyde 10. Reduction of the
aldehyde functions with lithium aluminiumhydride
yields alcohol 11.2 Amino-guanidine derivatives
(12, 21-26) were synthesised from the
Mannich-bases, (di) thiophene-diketones and
a-terthiophene-dialdehyde with amino-guanidine in
ethanol. Condensation of a-terthiophene-dialdehyde
10 with malonitrile in pyridine yields a
tetracyano derivative 13. 2-Cyano-?-terthiophene
14 was prepared from a-terthiophene and
chlorosulfonyl isocyanate in dichloromethane
followed by reaction with DMF. ?-Terthiophene-5-
carboxylic acid 15 was obtained from the
lithiated terthiophene quenched by carbon
dioxyde7.
Fig.3
Discussion We have synthesised and characterised
a series of novel thiophene and a-terthiophene
derivatives and they were tested in cell
proliferation assay on EGFR overexpressing tumour
cell line (A431) and six of them were found to be
active. One compound (12) had excellent
antiproliferative activity and two
amino-guanidine derivatives 21, 22 had cytotoxic
effect. Among the known PKC inhibitors
(dialdehyde 10, nitrile 14 and dihydroxyl 11
derivatives) only the parent a-terthiophene 4a
and its dialdehyde 10 showed antiproliferative
effect. Out of the 25 synthesised thiophene and
a-terthiophene analogs six had better than 80
proliferation inhibitory activity without
cytotoxic effect. The biological test results
showed that the disubstituted analogs had
stronger inhibitory activity than the
monosubstituted ones and basic moieties enhanced
their activity. Terthiophene-diaminoguanidine
derivative 12 has 99.80 inhibitory activity
against A431 cell culture, while unsubstituted
a-terthiophene 4a inhibited the proliferation
with 87.90. An interesting observation was that
the asymmetric terthiophene 4b has lower
inhibitory effect than symmetric terthiophene
4a. Current efforts in our laboratory are
directed toward the synthesis of new terthiophene
derivatives to increase the water solubility of
the molecule by coupling with hydrophilic
moieties, retaining the biological activity.
Another important viewpoint is the selectivity,
so we are going to test the active compounds in
other cell lines.
Reagents and conditions (a) LDA, abs. THF, -78
0C, 2 h DMF, 12 h, room temperature (b) LAH,
abs. THF, 4 h, room temperature (c)
aminoguanidine, EtOH, reflux temperature, 12 h
(d) malonitrile, pyridine, 100 0C, 2 h (e)
chlorosulfonyl isocyanate, abs.DCM DMF, 1 h,
room temperature (f) LDA, abs. THF, -78 0C, 2
h CO2, 12 h, room temperature
The Mannich-bases 6a and 6b were excellent
reagent to prepare diketone, hydrazone and
amino-guanidine derivatives. 6a was reacted with
aryl-aldehydes and NaCN in abs. DMF produced
diketone derivatives 3, 16-20. 21-26 were
synthesised from the Mannich-bases and (di)
thiophene-diketones with amino-guanidine in
ethanol. The hydrazones 27 and 28 were obtained
from 6a and aryl-hydrazins in EtOH at reflux
temperature.
Fig.4

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Reagents and conditions (a) aryl-aldehyde, NaCN,
abs. DMF, 12 h, room temperature (b)
aminoguanidine, EtOH, reflux temperature, 12 h
(c) phenyl-hydrazine, EtOH/HCl, reflux
temperature, 6 h
We gratefully acknowledge the contributions of
the following colleagues who support our work
János Pató, Richárd Schwáb, Edit Szabó, Ildikó
Szlágyi, István Varga