Robust synthesis of ortho-cyano-heteroarylamines, building blocks of ATP like kinase inhibitors

1. Robust synthesis of ortho-cyano-heteroarylamines, building blocks of ATP like kinase inhibitors Frigyes Wáczek1, 2*, Jenő Marosfalvi2, Zoltán Varga1, 2, Zsolt Székelyhidi1, 2, Péter Bánhegyi1, 2, Gyula Bencze1, Richárd Schwáb1, Edit Szabó1, Beatrix Németh1, György Kéri1, 2,, László Őrfi1, 2, 3 1 Cooperative Research Center, Semmelweis University, Budapest 5, Pf. 131, H-1367 Hungary, Tel/Fax: +36-1-3010613, E-mail: fwaczek@kkk.sote.hu 2 VICHEM Ltd., Herman O. u. 15, Budapest, H-1022 Hungary, 3 Department of Pharmaceutical Chemistry, Semmelweis University, Hőgyes E. u. 9, Budapest, H- 1092 Hungary *Corresponding author http://www.vichem.hu Introduction: The ATP binding sites of various enzymes are potential targets of enzyme inhibitory drugs, there is an increasing demand for ATP-like compounds. The structures of ATP analog inhibitors are mostly contain a condensed bicyclic heteroaryl moiety, within a pyrimidine ring part seems to be crucial. For the construction of pyrimidine ring in condensed bicyclic heteroaryl cores, the ortho-cyanoarylamines are very useful building blocks.1 In the present paper we show an effective and cheap method for the preparation of hundreds of ortho-cyanoarylamines. We produced more than 150 commercially unavailable molecules as proprietary intermediates which can serve as good starting points for the development of potential drug candidates. Biological activity of several derivates are also described. • Cooperative Research Center • Semmelweis University • Budapest, Hungary • http://www.webio.hu/kkk Published synthesis of the pyrazole building block and a simplified method of it’s preparation via enoyl chloride intermedier Traxler et al.1 have published the synthesis of 4-arylamino-3-phenyl-1H-pyrazolo[3,4-d]pyrimidines, as ATP-like kinase inhibitors (e.g. compound 2), via 5-amino–4-cyano–3-phenyl-1H-pyrazole key-intermediate 1. (Scheme1.) Synthesis of bioisosteric isoxazoles from common enoyl chloride intermediers We have found enoyl chloride intermediers of general formula A are usful for the synthesis of two aminoisoxazole carbonitrile isomers E and F(Scheme 3). 5-Aminoisoxazole-4-carbonitriles E, were obtained in the reaction of A with hydroxylamine in aqueous solution at neutral pH. The pH was adjusted by addition of adequate amount of NaOH, resulting deliberation of the free base, which is crucial in the first condensation step. The amino group as nucleophylic functional group reacts with the enoyl chloride in the first step followed by a fast ring-closure reaction yielding E without the chance of the isolation of intermediateB. Excess amount of base causes deprotonation of the hydroxyl function in the hydroxylamine, which has stronger nucleophylic property and reacts better with the enoyl chloride moiety of A, finally resulting the other isomer F. However the equilibrium may be pushed towards the formation of isomer F by addition of two equivalent NaOH, it is not suitable method for synthesis of pure product F, because about 20-30% of E isomer always contaminates the final product. An US patent from 1974 reported the application of hydroxyurea as protected hydroxylamine in the reaction with enoyl chlorides resulting 3-aminoisoxazole-4-carbonitriles of general formula F.6 The amino moiety is blocked in the hydroxyurea, preventing it’s reaction with enoyl chloride in the first step and directed the reaction via intermediers of general formula C. The protecting group leaves easily so it does not prevent the reaction of the amino moiety with the nitrile in the ring-closure, resulting F. The intermediate molecules of general formula C have not been separated in this case either. We produced more than 150 commercially unavailable molecules of general formula D, E and F by the reported method, these molecules are unique intermediates for drug development purposes. The antiproliferative effect of some derivates were proven in vitro on A431 cells and the apoptotic effect of some compounds were studied by flow cytometry (FACS). Scheme 1 (a) Triethyl orthoformate/100 oC (b) 3-Chloroaniline/ethanol/reflux (c) H2O/dioxane/reflux The preparation of compound 1 was reported starting from 2-(methylsulfanyl-phenyl-methylene)-malononitrile 9 and hydrazine hydrate with high yield.2 Although this reaction seems to be very practical for preparation of 1, the intermedier 9 is accessible through many reaction steps and complicated intermediates and it is finally derived from two simple starting materials: malononitrile and benzoyl chloride. (Scheme 2.) During the preparation of 1 the overall yields calculated for both starting materials individually are: 27,7% for 3 and 49.8% for 7 according to the literature 3-5 Scheme 3 Antiproliferative activity of the derivatives Searching for new antiproliferative agents we have modified compound 2 using our building block set described above. We have found taht the ring opened urea 12 and the isoxazolo-pyrimidine derivates of general formula 13 had antiproliferative effect in micromolar range on A431 cells. A431 epidermoid carcinoma cell (overexpressing EGFR) were harvested in standard cell-culture environment (37 °C, 5% CO2, 10% FCS). Proliferation assays were based on the Methylene Blue method.7 Cell viability was assessed after 6 and 48 hours. Efficacy (=low, non-specific toxicity + high rate of apoptosis induction) was judged on the basis of high early viability connected to the maximum viability-loss at 48h. Scheme 2 (a) Br2, KBr/water/5-10oC, (b) Cu/Benzene/reflux, (c) Iodosylbenzene /dichloromethane, (d) 2,4-Bis(methylthio)-2,4-dithioxo-1,3-dithia-λ5,λ5-diphosphetan/ benzene/80oC, (e) Benzene/r.t., (f)N2H4*H2O/MeOH/reflux, (g) Pyridine/0oC, (h) POCl3/reflux (i) N2H4*H2O/MeOH/reflux. Analysis of apoptosis induction based on propidium iodide staining Flow cytometry was used with propidium iodide staining, which is simple and cheap method for determination of membrane integrity and DNA content. A431 cells were treated with inhibitors for 24 hours prior to analysis.The ratio of Sub G1 area provides information about apoptotic events. Starting from the same simple molecules 3 and 7 we managed to prepare 1 through another reactive intermediate -chlorobenzylidene malononitrile 11, which can be obtained in two reaction steps. Although 11 is referred as precursor of other heterocyclic molecules, it has not been published as intermediate for synthesis of 5-amino–4-cyano–3-phenyl-1H-pyrazole 1 yet. This paper subjects our efforts for replacing intermedier 9 with the corresponding chlorobenzylidene intermedier 11 in the preparation of 1. This substitution of 2-methylsulfanyl moiety with 2-chloro function resulted the increase of reactivity while we obtained compound 10 with comparable yield in a significantly shorter reaction time in the ring-closure reaction. Our method is capable for synthesis of the molecule 1 via 11 from simple compounds 3 and 7 with an overall yield of 60% in 3 reaction steps. This method is independent from the processes and complicated intermediates of the reaction pathway referred in the literature: bromination of 3, coupling of 4 by copper powder forming 5, epoxidation of 5 and thiation of 7 resulting 8. DNA content Most of the cells are healthy and there is no sign of apoptotic events in SubG1 in DMSO control after 24h. 51% of the events in positive control (cycloheximide) are in SubG1 area after 24h. Both compounds 12 and 13a show very strong apoptotic effect reducing G1, S and G2 events below 20%. Conclusion:  In this paper we reviewed the chemistry of enoyl chloride intermediates which make them applicable as building blocks in the synthesis of biologically active compounds. We have shown that the applied method is capable for the synthesis of three different types of heteroaryl building blocks via common enoyl chloride intermediates. The process resulted the products from moderate to good yields. Commercially available, cheap chemicals were used in the synthesis like acyl chlorides, malononitrile and simple reagents. The elaborated synthesis also suffers from some limitations. It cannot be applied in the synthesis of unsaturated (e.g.: acryl methacryl) derivates. Although acryloyl and methacryloyl chlorides are commercially available, polymerization during the first acylation reaction diminished the yield of these derivates. The method is only limited by the feasibility of the acyl chlorides as starting-materials. We found these ortho-cyanoarylamines useful as starting materials in the synthesis of condensed heterocycles in the antiproliferative drug development field. Compound 12 (10 microM/24h) Compound 13a (10 microM/24h) Cycloheximide (10 microM/24h) SubG1 SubG1 SubG1 References: [1] Traxler, P. M.; Furet, P.; Mett, H.; Buchdunger, E.; Meyer, T.; Lydon, N.; J. Med. Chem. 1996, 39, 2285-2292 [2] Traxler, P. M.; Bold, G.; Frei, J.; Lang, M.; Lydon, N.; Mett, H.; Buchdunger, E.; Meyer, T.; Mueller, M. and Furet, P.; J. Med. Chem. 1997, 40, 3601-3616 [3] US 2794824 [4] Pryor, W., A., Govindan, C., K.; J.; Amer. Chem. Soc. 1981, 103, 25, 7681-7682 [5] Yousif, N. M.; Pedersen, U.; Yde, B.; Lawesson, S.-O.; Tetrahedron 1984, 40, 14, 2663-2670 [6] US 3816421 [7]Oliver M.H. et al. J. Cell. Sci. 92(3): pp. 513-18, 1989 The authors gratefully acknowledge the contributions of Dr. Ildikó Szilágyi and Mr.István Varga in the structure verification and the chemical analysis of the compounds.