Libraries from Libraries Approach to the Synthesis of Arylidene Oxindoles A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By KYLE JAMES KNISLEY B.S., Wright State University, 2011 2013 Wright State University WRIGHT STATE UNIVERSITY SCHOOL OF GRADUATE STUDIES December 5, 2013 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY KYLE JAMES KNISLEY ENTITLED Libraries from Libraries Approach to the Synthesis of Arylidene Oxindoles BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. Daniel Ketcha, Ph.D. Thesis Director David Grossie, Ph.D., Chair Committee on Final Department of Chemistry Examination College of Science and Mathematics Daniel Ketcha, Ph.D. Eric Fossum, Ph.D. Kenneth Turnbull, Ph.D. R. William Ayres, Ph.D. Interim Dean, Graduate School [Type text] Abstract Knisley, Kyle James. M.S., Department of Chemistry, Wright State University, 2013. Libraries from Libraries Approach to the Synthesis of Arylidene Oxindoles. A libraries from libraries combinatorial chemistry approach was employed to synthesize fluorinated derivatives of both oxindoles and isatins as potential pharmaceuticals or targeting agents for imaging purposes related to cancer or Alzheimer’s disease. Synthesis for these fluorinated derivatives are described by routes involving, either: a) N-alkylation of 5-substituted isatins followed by Wolff-Kishner reduction to the corresponding oxindoles and final Knoevenagel condensation with aryl aldehydes, or; b) Wolff-Kishner reduction of the isatins followed by condensation and finishing with the N-alkylation of the aldol products. In specific cases, a “click” reaction followed the N-alkylation of the aldol products to form the isatin 1,2,3-triazole which could be utilized to perform radiochemistry with a [18F]-radiolabel for the imaging of cancer. The strategy for the synthesis of such potential inhibitors was guided by SAR studies of peptide based inhibitors, as well as small-molecule inhibitors based upon the isatins scaffold. Previously, it was shown increasing functionality by adding 3 points of variability with the incorporation of an electron-withdrawing group such as a chlorine atom at the C-5 position allowed for increased potency of the oxindole derived inhibitors. Herein, a library of arylidene oxindoles was synthesized utilizing 3 points of variability with the incorporation of the electron-withdrawing group fluorine. Furthermore, a novel i [Type text] alternative synthesis was established for the creation of arylidene oxindoles which allowed for increased functionality through the incorporation of N-propargyl inhibitors. Finally, the ability to create N-propargyl compounds lead to the synthesis of isatin 1,2,3- triazoles was also explored for the possibility as potential imaging agents for cancer. ii [Type text] Table of Contents Page I. Introduction………………………………………………………………….1 A. Oxindole………………………………………………………………….2 B. Isatin………………………………………………………………….….10 C. Biological Background…………………………………………………13 D. Peptide Based Cell Death Inhibitors………………………………..…14 E. Small Molecule Approach……………………………………………...18 i. SmithKline Beecham……………………..…………………….18 ii. Washington University……………………………………..…..24 F. Small Molecule Imaging Agents for Cancer…...……………………..27 G. Alzheimer’s Disease..…………………………………………………...46 II. Aims and Background to Research……………………...………...………52 A. WSU Approach……..…………………………………………………..52 III. Results and Discussion……………………………………………………...61 A. N-Alkylation of Isatins……………..…………………………………...61 ii. N-Alkylation of Isatin Utilizing DBU..………………………...65 iii. N-Alkylation of Isatin Utilizing KF/Al2O3…..………………..70 B. Reduction of Isatin…………………..………………………………….71 i. WSU Wolff-Kishner Reduction of Isatins to Oxindoles…..…73 C. Aldol Condensation……………………………..………………………76 iii [Type text] D. Alkylation of Benzylidene Oxindoles...………………..………………93 E. Alternative Synthesis Route…………………………………………...95 F. Click Chemistry…………………………………………………….....107 G. WSU Approach to Click Chemistry………………………………....111 H. NMR Analysis………………..………………………………………..113 I. NMR Analysis of N-Substituted Isatins……………………...……...113 J. NMR Analysis of 5-Fluorooxindoles and N-Substituted 5- Fluorooxindoles……………………………………………………….117 K. NMR Analysis of 5-Fluoro-3-substituted Indolin-2-ones………......120 L. Summary and Conclusions………...…………………………………121 M. Experimentals………………………………………………………….124 N. References……………………………………………………………...193 iv [Type text] List of Figures/Schemes Page Figure 1- Structural Design Features of Benzylidene Oxindoles……………………54 Scheme 1- Reaction Manifold………………………………………………………….54 Figure 2- 5- Fluoroisatin…………………………………………………………113, 115 Figure 3- 1H NMR Spectrum of 5-fluoroisatin…………………………………..…..114 Figure 4- 13C NMR Spectrum of 5-fluoroisatin……………………………………...115 Figure 5- 13C NMR Spectrum of N-benzyl-5-fluoro-indolin-2,3-dione…………....116 Figure 6- 13C NMR Spectrum of N-2,6 difluorobenzyl-5-fluoro-indolin-2,3- dione……………………………………………………………………………………117 Figure 7- 13C NMR Spectrum of 5-fluoro-indolin-2-one………..…………………..118 Figure 8- 13C NMR Spectrum of N-benzyl-5-fluoro-indolin-2-one………….……..119 Figure 9- 13C NMR Spectrum of N-2,6-difluorobenzyl-5-fluoro-indolin-2-one…..120 Figure 10- 1H NMR of 5-fluoro-3-(2,6-difluorobenzylidene)-indolin-2-one..……..122 v [Type text] List of Tables Page A. Table 1: N-alkylation of 5-substituted isatin derivatives via DBU…66 B. Table 2: Alkylation of 5-substituted isatin derivatives via KF/Al2O3………………………………………………………….…….70 C. Table 3: Wolff Kishner Reduction of N-alkylated isatin derivatives………………………………………………………………74 D. Table 4: 3-Substituted-Indolin-2-ones from Knoevenagel Condensations…………………………………………………………..79 E. Table 5: N-(2,6-Difluorobenzyl)-5-fluoro-3-Substituted-Benzylidene- Indolin-2-one……………………………………………………………82 F. Table 6: N-(Benzyl)-5-fluoro-3-Substituted-Benzylidene-Indolin-2- ones……………………………………………………………………....85 G. Table 7: N-(3,5-Difluorobenzyl)-5-fluoro-3-Substituted-Benzylidene- Indolin-2-ones…………………………………………………………...89 H. Table 8: N-(4-Methoxybenzyl)-3-Substituted-Benzylidene-Indolin-2- ones………………………………………………………………………92 vi [Type text] I. Table 9: 3-Substituted-benzylidene-indolin-2-ones…………………..97 J. Table 10: 5-Chloro-3-substituted benzylidene-indolin-2-ones……....99 K. Table 11: Arylidene Oxindoles via N-Alkylation of Benzylidene Oxindole ……………………………………………………………….103 L. Table 12: N-Propargyl-3-substituted benzylidene-indolin-2-ones....105 M. Table 13: Isatin Triazoles……………………………………………..112 vii [Type text] Acknowledgements I would like to give special thanks to Dr. Ketcha for supporting me throughout my time at Wright State University. I appreciate all the support and opportunities you have provided for me and I couldn’t have asked for a better advisor or person to work with over the past two years. I would also like to give thanks to all the Chemistry faculty members at Wright State as well as supporting staff, friends, and visitors. viii [Type text] Introduction In recent years the drug discovery process has relied to some extent on the precepts of combinatorial chemistry1 as a means of rapidly synthesizing and evaluating diverse libraries of compounds for biological activity. Such compound libraries are centered about scaffolds which can be defined as the “core portion of a molecule common to all members of a combinatorial library.”2 Certain chemical structures (often polycyclic heterocycles) have been found to be particularly attractive scaffolds for drug discovery libraries as they are often capable of binding as ligands to multiple, unrelated classes of protein receptors and have been defined as privileged structures.3 One such family of privileged structures includes the benzo-fused nitrogen heterocycles, including indole (1)4 and its oxidized congeners isatin (2) and oxindole (3). It is generally appreciated that libraries developed around such privileged structures should “yield medicinally active compounds with high hit rates at significantly reduced library size compared to large classical libraries obtained from combinatorial chemistry efforts based on non-privileged templates.”5 As this thesis relates to the construction of fluorinated derivatives of both oxindoles and isatins as potential 1 [Type text] pharmaceuticals or targeting agents for imaging purposes related to cancer or Alzheimer’s disease, a brief overview of the diverse biological activities of these heterocycles is presented herewith. Oxindole Oxindole (indolin-2-one, 3) is a reduced derivative of the isatin family and was first synthesized by Baeyer at the end of the 19th century through the reduction of isatin.6 The chemistry and synthesis of oxindole was last reviewed by Sumpter in 1945.7 Given the biological activity of this heterocyclic scaffold, much recent work has been devoted to rapidly generating diversity by taking advantage of the reactive C-3 ketone carbonyl group of isatin precursors in multicomponent reaction processes so as to afford the corresponding spirocyclic oxindoles.8 Although the broad range of biological activities exhibited by oxindoles warrant their inclusion into the classification of privileged scaffolds, perhaps their most significant role is that of protein kinase inhibitors.9 Arylidene oxindoles were among the first structures identified as receptor tyrosine kinase (RTK) inhibitors by SUGEN in 1998, wherein it was found that: (1) 3- [(pyrrole)methylidenyl]indolin-2-ones are highly specific against the VEGF (Flk-1) RTK; (2) 3-(substituted benzylidenyl)indolin-2-ones