1. Synthesis of thiophene and α-terthiophene derivatives with antiproliferative activity Zsolt Székelyhidi a *, János Pató b, László Őrfi a,b,c, Frigyes Wáczek a, Péter Bánhegyi a, Gyöngyi Bökönyi d, Edit Z. Szabó d, Edit Várkondi a, Ákos Pap a, Richárd Schwáb a and György Kéri a,b,d a Cooperative Research Centre, Semmelweis University, Rippl-Rónai u. 37., 1062 Budapest, Hungary b Vichem Chemie Ltd., Hermann u. 15., 1022 Budapest, Hungary c Dept. of Pharmaceutical Chemistry, Semmelweis University, Hőgyes u. 9., 1092 Budapest, Hungary d Peptide Biochemistry Research Group, Hung. Acad. Sci - Semmelweis Univ., Puskin u. 9., 1088 Budapest, Hungary *Tel.: +36-1-3010614; Fax: +36-1-3010613; szekelyhidi@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 kinases 1. Thousands of compounds around several scaffolds have been developed as potential kinase inhibitory drugs. A few terthiophenes are known as PKC inhibitors 2,3 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, β-cellulin and transforming growth factor-α 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 We have tried the synthetic route described in the literature to prepare α-terthiophene. According to this method succinyl chloride 1 was reacted with thiophene 2 in the presence of aluminium chloride in dichloromethane at 0 0 C to obtain the dithiophene-1,4-diketone 3 in 80% yield. Thionation using Lawesson`s 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 Mannich–bases 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. 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) P 2 S 5, NaHCO 3, THF/hexane=3/1, 12 h, room temperature Reagents and conditions: (a) AlCl3, DCM, 0 0C; (b) Lawesson`s reagent Fig.2  -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 α-terthiophene-dialdehyde with amino-guanidine in ethanol. Condensation of α- terthiophene-dialdehyde 10 with malonitrile in pyridine yields a tetracyano derivative 13. 2-Cyano-  -terthiophene 14 was prepared from α-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 dioxyde 7. 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 Fig.3 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. 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 Fig.4 Biological test Human A431 epidermoid carcinoma cells were cultured in DMEM (Dulbecco’s 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 37˚C 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/C*100). 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) > 80%. IC50 values were generated from IC50 graphs. Discussion We have synthesised and characterised a series of novel thiophene and α-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 α-terthiophene 4a and its dialdehyde 10 showed antiproliferative effect. Out of the 25 synthesised thiophene and α-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 α-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. References Janet Dancey; Edward A. 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Bioorg.Med.Chem.Lett. 1999, 9, 3411-3416 We gratefully acknowledge the contributions of the following colleagues who support our work: János Pató, Richárd Schwáb, Edit Szabó, Ildikó Szlágyi, István Varga