1. 5,6,7,8-Tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-4-yl-amine Derivatives as Potent and Selective, ATP Side Directed Inhibitors of the EGF-Receptor Protein Tyrosine Kinase Péter Bánhegyi a *, László Őrfi a,b,c, Frigyes Wáczek a, Zsolt Székelyhidi a, Gyöngyi Bökönyi d, Edit Z. Szabó d, Edit Várkondi 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; banhegyi@kkk.sote.hu Introduction Protein tyrosine kinases (PTK) play a fundamental role in signal transduction pathways. 4 Deregulated PTK activity has been observed in many proliferative diseases (e.g. Cancer, psoriasis, restenosis, etc.). 1 Tyrosine kinases are therefore attractive targets for the design of new therapeutic agents. The PTK`s can be divived into subgroups which have similar structural organization and amino acid sequence similarity within their kinase domains. 2 The family of the epidermal growth factor receptor (EGF-R) PTK belongs to the largel class of the transmembrane growth factor receptor PTK`s. This EGF-R family contains four members, the EGF-R kinase (c-erb B-1 gene product), the p185 erb B2 (c-erb B-2 gene product), and the recently identified c-erb B-3 and c-erb B-4 gene products. EGF-R and its ligands (EGF, TGF-α) have been implicated in numerous tumors of epithelial origin (e.g. squamous cell carcinoma: breast, ovarian, NSC lung cancer; etc.) 1,3 and proliferative disorders of the epidermis such as psoriasis. 4 Inhibitors of the EGF-R PTK could therefore have great therapeutic potential in the treatment of malignant and nonmalignant epithelial deseases. Due to the involvement of tyrosine kinases in many signal transduction pathways, it will be important to develop inhibitors with high selectivity at the enzyme level. Fig.1 Compounds of general structure 8 and 9 can be obtained from common starting material 2-amino-4,5,6,7-tetrahydro- benzo[b]thiophene-3-carbonitrile (3), which was preparated from cyclohexanone, malonitrile and sulphur by Gewald reaction. Ring-closed derivates 4 and 5 were preparated by heating of 3 in formic acid or in cyclopropanecarbonic acid. The pyrimidone intermediers 4, 5 were refluxed in phosphoroxychloride yielding active imidoyl chloride derivates 6, 7 which were reacted with the correspoding aniline resulting the desired compound families. Fig2 (a) Gewald reaction (Gewald, K.; Schinke, E.; Böttcher, H. Chem. Ber. 1966, 99, 94 ) (b) Stirred in Cyclopropanecarboxylic acid, at 120 o C, for 2 hours (c) Refluxed in 80 % Formic acid, for 2 hours (d) Refluxed in phosphoroxychlorid for 1 hour (e) Stirred with the correspoding aniline in Ethanol Fig.3 The following 5,6,7,8-tetrahydro-benzo[4,5]thieno [2,3-d]pyrimidin-4-ylamine compounds of general structure 8, 9 were designed and preparated. Fig3 Kinase assay: Materials – All compounds were dissolved in DMSO and diluted in reaction buffer (500 mM Hepes pH 7.4, 200 mM MgCl 2, 1 mM MnCl 2, 2 mM Na 3 VO 4 ) for tyrosine kinase assay. Single concentration of compounds (10 μM) containing 1% DMSO were tested in duplicates. For tyrosine kinase assay, 50 ng recombinant EGFR enzyme (ProQinase) and 30 μg Poly(Glu,Tyr) substrate (Sigma) was used. Phosphorylated substrate was detected by HRP-conjugated anti-phosphotyrosine antibody and OPD (Orthophenildiamine) (Sigma). and the reference EGFR inhibitor was used as positive control. For assaying tyrosine kinase function a modified protocol of Sigma (PTK101) was used. 96-well plates were coated with Poly(Glu,Tyr) substrate in PBS at 37˚C overnight, then washed with PBS containing 0,05% Tween once. Then EGFR and ATP in reaction buffer were added and incubation at 37˚C for 30 minutes followed. Plates were washed 5 times and HRP-conjugated anti-phosphotyrosine antibody was added at a dilution of 1:1000. After 30 minutes of incubation at 37˚C, plates were washed 5x and OPD was added along with H 2 O 2. Reaction was stopped with H 2 SO 4 and plates were read by ELISA reader at 490 nm. Statistics – Results are expressed as OD of the percentage of Treated / Control wells (T/C%). Discussion We have synthesised and characterised two series of novel 5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-4-yl-amine derivates, and they were tested in cell proliferation assay on EGFR overexpressing tumour cell line (A431) and against EGF-R PTK inhibition. Five of them were found to be active on the proliferation assay (8a, 8d, 8f, 8h, 9g) and four on the EGF kinase assay (8a, 8d, 8g, 8h). We found the cyclopropyl moiety at 2 position (general formula 9) ruind the inhibitory activity of the compounds in both assays. Fig4 Current efforts in our laboratory are directed toward the synthesis of new 5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3- d]pyrimidin-4-yl-amine derivatives (e.g. the unsaturated ring system, another R1 groups, etc.) to increase the biological activity. Another important viewpoint is the selectivity, so we are going to test the active compounds in other cell lines. References 1.Aaronson, S. A. Growth Factors and Cancer. Science, 1991, 254, 1146-1152 2.Hanks, S. K., Quinn, A., M., Hunter, T. The protein kinase family: conserved features and phylogeny at the catalytic domains. Science, 1988, 241 42-52 3.Ulrich, A., Schleissinger, J. Signal Transduction by Receptors with Tyrosine KinaseActivity. Cell 1990, 61, 203-212 4.Elder, J. T.; Fisher, G. J.’ Lindquist, P. B.; Bennett, G. L.; Pittelkow, M. R.; Coffey, R. J.; Ellingsworth, L; Derynck, R.; Voorhees, J. J. Overexpression of transforming growth factor α in psoriatic epidermis. Science 1989, 243, 811-814 5.Peter M. Traxler, Pascal Furet, Helmut Mett, Elisabeth Buchdunger, Thomas Meyer, Nicholas Lydon> J. Med. Chem, 1996, 39, 2285-2292 6.H. D. Hollis Showalter et all. J. Med. Chem, 1999, 42, 5464-5474 We gratefully acknowledge the contributions of the following colleagues who support our work: Ildikó Szilágyi, István Varga, Ferenc Jaczina Reagents and conditions: Peter Traxler et all published 5 the 4-(phenylamino)pyrrolopirimidin derivates as ATP site directed inhibitors of the EGF-R PTK. Aromatization of the tetramethylene ring attached to the pyrrole ring (X=(CH 2 ) 4 -> (CH) 4 ) incrased the potency. The benzo[4,5]thieno[3,2-d]pyrimidin-4-yl-phenyl derivates were described by D. Hollis Showalter et all 6 as efective EGF-R PTK inhibitors. (Fig.1) A couple of 5,6,7,8-tetrahydro-benzo[4,5]thieno [2,3-d]pyrimidin-4-ylamine compounds of general structure 8 and 9 were designed and preparated starting out from the structures of the most active tricyclic EGF-R PTK inhibitor molecules 1 and 2. Present poster is aiming the publication of the synthesis and biological data of these molecules. Biological tests Proliferative assay: 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. Compound Remaining activity at 10 uM 8a40% 8b100% 8c64% 8d44% 8e100% 8f75% 8g44% 8h41% 9a71% 9b100% 9c100% 9d85% 9e90% 9f95% 9g99% Reference110% Reference262% Fig.2 Synthetic procedures Waters Alliance LC system, equipped with Micromass Qvadrupole MS detector was used for the purity, chemical identity analysis. The chemical identities of molecules were also checked by 1 H NMR spectra, which were made on Bruker 300 MHz NMR spectrometer in DMSO-d6. Fig.4