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Margarta VOJTIKOV under the direction of: Assoc. Prof. Andrej BOH Dr. Gilles HANQUET 27 th September 2013, Strasbourg

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Summary 1) Introduction 2) Angiogenesis 3) Aim of the project 4) Study towards target ynamides 5) Azides 6) Click Chemistry 7) Biological assays 8) General conclusion 2 IntroductionAngiogenesis Aim of the project YnamidesAzides Click Chemistry Biological assays General conclusion

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Introduction WHO: Cancer is uncontrolled growth and spread of cells Cancer leading cause of mortality 7.6 million dead people in 2008 (13 % of all deaths) neccessity to develop new active compounds for cancer treatment 3

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Angiogenesis 4

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Aim of the project PDB: 1Y6A tyrosine kinase complex prepared in low ca 10 % yield over 5 steps sensitive N-aryloxazole-2-amine group 5 Harris, P. A.; Cheung, M.; Hunter, R. N.; Brown, M. L.; Veal, J. M.; Nolte, R. T.; Wang, L.; Liu, W.; Crosby, R. M.; Johnson, J. H.; Epperly, A. H.; Kumar, R.; Luttrell, D. K.; Stafford, J. A. J. Med. Chem. 2005, 48, 1610.

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1,3-oxazole / 1,2,3-triazole replacement (me-too or me-better) exchange of heterocyclic core: activity and/or selectivity higher stability synthetic feasibility better physical and chemical properties for bioavailability lower toxicity inhibitor novelty 6

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Predicted 1,2,3-triazoles interaction analysis, molecular modelling, docking 7 triazolic analogues of PDB : 1Y6A Discovery Studio Visualizer 3.5 software Drug like properties prediction toolkit Molinspiration 7 http://www.rcsb.org/pdb/home/home.do http://accelrys.com/products/discovery-studio/visualization-download.php Molinspiration Property Calculation Service http://www.molinspiration.com/cgi-bin/properties 1,4-regioisomers

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docking scores and ligand possess in VEGFR2 are less interesting significantly higher price of ruthenium catalyst 8 1,5-regioisomers

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Retrosynthetic approach 9

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Ynamides Literature background 10 chemistry of ynamides exploded in last decade heteroatom-substituted alkynes ynamines unstable, the firts report in 1892 ynamides right balance between stability and reactivity Bode, J. Liebigs Ann. Chem. 1892, 267, 268. DeKorver, K. A.; Li, H. ; Lohse, A. G. ; Hayashi, R. ; Lu, Z. ; Zhang, Y. ; Hsung, R. P. Chem. Rev. 2010, 110, 5064. Evano, G. ; Jouvin, K.; Coste, A. Synthesis 2013, 45, 17.

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Ynamides Literature background - preparation first synthesis of electron deficient ynamines Viehe, 1972 Zhdankin & Stang, 1994 synthesis of ynamines using alkynyl iodonium triflate / tosylate salts 11 Janousek, Z. ; Collard, J. ; Viehe, H. G. Angew. Chem. Int. Ed. 1972, 11, 917. Murch, P.; Williamson, B. L.; Stang, P. J. Synthesis 1994,1255. Kitamura, T.; Tashi, N.; Tsuda, K.; Fujiwara, Y. Tetrahedron Lett. 1998, 39, 3787.

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expansion of chemistry of ynamides Witulski, Rainer 12 Witulski, B.; Stengel, T. Angew. Chem. Int. Ed. 1998, 37, 489. Witulski, B.; Gmann, M. Synlett 2000, 1793. Witulski, B.; Stengel, T. Angew. Chem. Int. Ed. 1998, 38, 2426. Witulski, B.; Stengel, T.; Fernandez-Hernandez, J.M. Chem. Commun. 2000, 1965. Witulski, B.; Alayrac, C. Angew. Chem. Int. Ed. 2002, 41, 3281. Rainier, J. D.; Imbriglio, J. E. J. Org. Chem. 2000, 65, 7272. Rainier, J. D.; Imbriglio, J. E. Org. Lett. 1999, 1, 2037.

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Elimination protocols Zemlicka, 1994 preparation of ynamides via lithium-halogen exchange Brckner, 2000 preparation of ynamides from formamides via lithium- halogen exchange 13 Brckner, D. Synlett 2000, 1402. Brckner,D. Tetrahedron 2006, 62, 3809. Yang, B. H.; Buchwald, S. L. J. Organomet. Chem. 1999, 576, 125.

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Direct N-alkynylation using bromoalkynes Buchwald copper catalyzed (CuSO 4. 5H 2 O, CuI, Cu 2 O, Cu(OAc) 2 N-arylations of amides Tam, 1996 improvement of the methodology using KHMDS Skrydstrup, 2008 second generation of Hsungs protocol mild base - K 3 PO 4 or K 2 CO 3 higher yields 52 91 % 14 J. Am. Chem. Soc. 2002, 124, 7421. Tetrahedron 2006, 62, 3823 J. Org. Chem. 2008, 73, 9447

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Ynamides Literature background - utilization 2013 Evano modular indole synthesis 2006 Cintrant - -addition of ynamides hydrostannylation 2006 Hsung et al. Lindlar type hydrogenation of ynamides 15 Org. Lett. 2013, 15, 3122. Tetrahedron Lett. 2006, 47, 3139 J. Org. Chem. 2006, 71, 4170.

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Tam 2006 ruthenium catalyzed [2+2]-cycloaddition of alkenes (bi- and tricyclic) with ynamides Saito 2012 total synthesis of (-)-Herbindoles A-C - [2+2+2]-cycloaddition Click chemistry K. B. Sharpless 2001 16 Riddell, N.; Villeneuve, K.; Tam, W. Tetrahedron 2005, 7, 3681. Villeneuve, K.; Riddell, N.; Tam, W. Tetrahedron 2006, 62, 3823. Saito, N.; Ichimaru, T.; Sato, Y. Org. Lett. 2012, 14, 1914.

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Ynamides - Proposed synthesis 17

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Preparation of model ynamide A) Corey-Fuchs approach published by Brckner crucial step - selection of EWG (Boc, Piv, Ts) B) Bestmann-Ohira reaction one step synthesis, all the attempts failed 18 Brckner, D. Synlett 2000, 1402. Brckner,D. Tetrahedron 2006, 62, 3809.

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Preparation of target ynamide Pathway A: via Corey-Fuchs approach problematic preparation of Corey-Fuchs precursor 19

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Pathway A: formylation in good yield tosylation - p-TsCl, without or with (Et 3 N, pyridine, NaH, n-BuLi) base Pathway B: tosylation in good yield formylation HCOOH + DCC, CDI; BtCHO; HCOOEt + base; Vilsmeier-Haack formylation; Eschenmoser salt; acetic-formic anhydride + base 20

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21 Application of Corey-Fuchs approach on tosylated N-formamide.

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Pathway C: Transformation of trichloroacetamides to ynamides Speziale, Smith - 1962; Himbert, Regitz 1972 key step conversion of trichloroenamine to lithiated ynamine 22 Speziale, A. J.; Smith, L. R. J. Am. Chem. Soc. 1962, 84, 1868. Himbert, G.; Regitz, M. Chemische Berichte 1972, 105, 2963.

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Pathway D: Direct N-alkynylation of arylamines using iodonium triflate salts Preparation of triflate salt 23 Tanaka, K.; Takeishi, K. Synthesis 2007, 18, 2920. Kerwin, S.; Nadipuram, A. Synlett 2004, 1404. potentialy explosive

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using bromoalkynes 2 possible reagents: Tams protocol -COOMe EWG small yield of alkynylation step 28 %; overall yield 26 % (3 steps) 24 Pathway D: Direct N-alkynylation of arylamines low boiling point

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Skrydstrup protocol 2 key ynamides prepared using COOMe - Overall yield 88 % over 3 steps alkynylation step 97 % using Boc- EWG overall yield 52 % over 3 steps 25 Skrydstrup., T. ; Dooleweerdt, K. ; Birkedal, H. ; Ruhland, T. J. Org. Chem. 2008, 73, 9447.

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Azides synthesis of several aromatic azides as partners for Click Chemistry discovered 140 years ago by Grie polar mesomeric structures Preparation: from diazonium salts 26 Grie, P. Philos. Trans. R. Soc. London 1864, 13, 377. Grie, P. Justus Liebigs Ann. Chem. 1865, 135, 131. Capitosti, S. M.; Hansen, T. P.; Brown, M. L. Org. Lett. 2003, 5, 2865.

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SN aromatic SN aromatic from non-activated aromatic halides Ma, Zhu 2004 copper-catalyzed reaction, EtOH / water Liang 2005 mild conditions 27 Zhu, W.; Ma, D. Chem. Comm. 2004, 888. Andersen, J.; Madsen, U.; Bjrkling, F.; Liang, X. Synlett 2005, 14, 2209.

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synthesis of appropriate azides azidation and Suzuki-Miyaura cross-coupling key steps 28 Preparation of target azides

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azide with ortho-substituted pyridyl cycle 57 % overall yield over 2 steps azide with meta-substituted pyridyl cycle 45 % overall yield (2 steps) 29 Trokowski, R.; Akine, S.; Nabeshima, T. Dalton Trans. 2009, 46, 10359. Li, W.; Nelson, D. P.; Jensen, M. S.; Hoerrner, R. S.; Cai, D; Larsen, R. D. Org. Synth. 2005, 11, 393.

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C-H palladium activated acetoxylation recently developed method for heteroatom-directed functionalization Ph(IOAc) 2, Pd(OAc) 2 in acetanhydride and benzene 30 Daugulis, O.; Zaitsev, V. G. Angew. Chem., Int. Ed. 2005, 44, 4046.

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Preparation of MOM- protected azidobiaryl V.47d and unsuccessful preparation of hydroxylated azidobiarym V.40d 31 Doyagez, E. S. Synlett 2005, 10, 1636. Soni, A.; Dutt, A.; Sattigeri, V.; Cliffe, I. A. Synth. Commun. 2011, 41, 1852.

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Pyrrole azide V.43 traces after Suzuki coupling azidation not performed 32 Gu, Z. ; Zakarian, A. Org. Lett. 2010, 12, 4224. Morrison, M. D. ; Hanthorn, J. J. ; Pratt, D. A. Org. Lett. 2009, 11, 1051.

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Proposed new retrosynthetical approach Prepared 2 new azides V.78a and V.78b 33 14% over 3 steps 19% over 4 steps

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Preparation of urea azide V.42 Overall yield 37 % over 7 steps 34 Kotha, S.; Shah, V. R. Eur. J. Org. Chem. 2008, 1054. Ganesh, T.; Thepchatri, P.; Du, L. L. Y.; Fu, H; Snyder, J. P.; Sun, A. Bio. Med. Chem. Lett. 2008, 4982. Chandrappa, S.; Vinaya, T.; Ramakrishnappa, T.; Rangappa, K. S. Synlett 2010, 3019. Deng, Q. H.; Wang, J. C.; Xu, Z. J.; Zhou, C. Y.; Che, C. M. Synthesis 2011, 18, 2959.

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Preparation of pyrimidine azide V. 42 2 synthetical pathways performed 35 Yaziji, V,; Rodriguez, D.; Guierrez-de-Terran, H.; Coehlo, A. ; Caamano, O. ; Garcia-Mera, X. ; Brea, J. ; Loza, M. I. ; Cadavid, M. I. ; Sotelo, E. J. Med. Chem. 2011, 54, 457. Chang, L. C. W.; Ijzerman, A. P.; Brussee, J. Oct. 1, 2004, United States Patent US 2007/0032510. Ye, C.; Gao, H.; Boatz,Drake, G. W.; Twamley, B.; Shreeve, J. M. Angew. Chem. Int. Ed. 2006, p. 7262.

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Click Chemistry K. B. Sharpless, 2001 Requirements: modular and wide in scope higly efficient and give high yields no or inoffensive by-poducts stereospecific readily available reagents no solvents or benign solvents simple purification non-chromatographic techniques 36 K.B. Sharpless Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004. Becer, C. R.; Hoogenboom, R.; Schubert, U. S. Angew. Chem. Int. Ed. 2009, 48, 4900.

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Huisgen thermal 1,3-dipolar cycloaddition of alkynes and azides Click Chemistry CuAAC, RuAAC MW accelerated Click Chemistry Cintrat and Ijsselstijn, 2006 Click Chemistry with ynamides 2004 Eycken 3-component MW accelerated Click chemistry reaction 37 Huisgen, R. Angew. Chem., Int. Ed. 1963, 2, 633. Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.; Sharpless, K. B.; Fokin, V. V. J. Am. Chem. Soc., 2005, 127, 210. Cintrat, J.C.; IJsselstijn, M. Tetrahedron 2006, 62, 3837. Appakkuttan, P.; Dehaen, W.; Fokin, V. V.; Eycken, E. V. Org. Lett. 2004, 6, 4223.

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Preparation of In Silico predicted triazoles via CuAAC in mild conditions using the prepared azides and ynamides Classical retrosynthetical approach 38

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Preparation of triazole III.26 with MeCOO- protecting group 40 Problematic deprotection of EWG EntryConditionsResults 11 M KOH in MeOH, rt, overnight Starting material VI.12 + products of decomposition VI.14 + VI.15 21 M KOH in MeOH, rt, 20 minStarting material VI.12 31 M KOH in MeOH, reflux, 20 minProducts of decomposition VI.14 + VI.15 4 0.5 M KOH in ethylene glycol + water, rt, 20 min Starting material VI.12 50.5 M KOH in ethylene glycol + water, reflux, 20 min Starting material VI.12 + products of decomposition VI.14 + VI.15

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with Boc- protecting group 41 EntryConditionsResults 15 equiv TBAF, THF, rt, overnightStarting material VI.16 25 equiv TBAF, THF, reflux, 30 minProducts of decomposition 312 M HCl / EtOAc = 1 / 2.3, rt, 1 hour Expected product III.26 + Starting material VI.16 + Products of decomposition 4TFA, rt, 1 hExpected product III.26 + products of decomposition [.[. Coleman, C. M.; OShea; D. F. J. Am. Chem. Soc. 2003, 4054. Englund, E. A.; Gopi, H. N.; Appella; D. H. Org. Lett. 2004, 213

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Alternative retrosynthetical approach preparation of triazole III.24 42

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preparation of triazole III.23 problem with solubility and purification of the resulting triazole 43 not prepared

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44 Prepared triazoles

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Biological assays IC 50 biological activity radiometric protein kinase assay 3 active 2 not active 45

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redocking biological activities not corresponded to docking score newer version Dock 3.6 with PDB : 1Y6A triazolic compounds much more worse binding energies than oxazolic predicted energies of triazoles ca 21 % less favorable compare to oxazolic isosters 46

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influence of isosteric oxazole / triazole replacement oxazolic core in PDB : 1Y6A surronded by liphophilic amino acid residues less favourable to bind triazole dipole moment 47 Discovery Studio http://accelrys.com/products/discovery-studio/visualization-download.php (visited 2 nd August 2013)

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General conclusion study towards preparation of target ynamides prepared 7 azides suitable for Click Chemistry approach 48

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prepared 5 desired 1,2,3-triazoles 3 active VEGFR-2 inhibitors 2 not active inhibitors 49

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Acknowledgement To my supervisors Assoc. Prof. Andrej BOH Dr. Gilles. HANQUET To members of jury my co-workers from ECPM and Faculty of Natural Sciences in Bratislava Financial support Comenius University COST STSM 0602 French Embassy in Bratislava my parents and all my friends 50