INVESTIGATIONS INTO THE CHEMOPREVENTIVE POTENTIAL OF STILBENES, INDOLINONES AND ISOINDIGOS: SYNTHESIS AND MODE OF ACTION STUDIES ZHANG WEI (B.Sc., SOOCHOW UNIVERSITY) NATIONAL UNIVERSITY OF SINGAPORE 2008 INVESTIGATIONS INTO THE CHEMOPREVENTIVE POTENTIAL OF STILBENES, INDOLINONES AND ISOINDIGOS: SYNTHESIS AND MODE OF ACTION STUDIES ZHANG WEI (B.Sc., SOOCHOW UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgements With my deepest gratitude and delight, I would like to dedicate my acknowledgment to my supervisor, Assoc. Professor Go Mei Lin for her constant encouragement and guidance. Without her consistent and illuminating instruction, this thesis could not have reached its present form. Secondly, I would like to thank Professor Loh Teck Peng and his postgraduate students Zhao Yu Jun and Shen Zhi Liang for invaluable discussions and suggestions for organic synthesis. I want to thank Oh Tang Booy and Ng Sek Eng for providing technical assistance. Thirdly, I would like to thank all technical and research staffs in department of pharmacy for their help and support. In addition, I want to thank all postgraduate students and FYP students in Medicinal Chemistry Research Lab, Liu Xiao Ling, Lee Chong Yew, Leow Jo Lene, Sim Hong May, Nguyen Thi Hanh Thuy, Wee Xi Kai, Wee Kiang Yeo, Liu Jian Chao, Tee Hui Wearn and Suresh Kumar Gorla. Many thanks to my friends Reng Yu Peng, Chen Wei Qiang, Li Cheng, Ling Hui, Wang Chun Xia. I am gratefully acknowlege the research scholarship from National University of Singapore. Last but not least, my thanks would go to my beloved family for their loving considerations and great confidence in me all through these years. I wish to give special thanks to my wife Liu Xiao Hong for her consistent encouragement and support, to allow me to finish this thesis. I Table of Contents Acknowledgements I  Table of Contents II  Publications and Conferences VI  Summary . VII  Chapter Introduction . 1  1.1.  Chemoprevention of Cancer . 1  1.2.  Potential Mechanisms of Chemoprevention 3  1.3. Induction of NQO1 as a Chemopreventive Strategy 4  1.4.  Regulation of NQO1 by the ARE/XRE and Keap1/Nrf2/ARE Pathways . 6  1.5.  Monofunctional and Bifunctional Inducers of Phase II Enzymes 7  1.6.  Stilbenes as Lead Structures for Chemopreventive Activity . 9  1.7.  Statement of Purpose 11  Chapter Design and Synthesis of Target Compounds 16  2.1.  Introduction 16  2.2.  Methoxystilbenes . 16  2.2.1  Rationale of drug design . 16  2.2.2. Chemical considerations 18  2.2.3.  Assignment of configuration . 23  2.2.4.  Experimental methods 24  2.3.  3-Substituted Indolin-2-ones 29  2.3.1.  Rationale of drug design . 29  2.3.2.  Chemical considerations . 33  2.3.3.  Assignment of configuration . 39  2.3.4.  Experimental methods 44  II 2.4.  Summary 47  Chapter Induction of NQO1 Activity by Methoxystilbenes . 48  3.1.  Introduction 48  3.2.  Experimental Methods . 48  3.2.1.  Materials . 48  3.2.2. Cell lines . 49  3.2.3. MTT assay for determination of cell viability 49  3.2.4. Determination of NQO1 activity 50  3.2.5. Quenching of ABTS radical cation . 51  3.2.6.  Measurement of 7-ethoxyresorufin O-deethylase (EROD) activity in Hepa1c1c7 cells 52  3.2.7.  In silico determination of log P . 53  3.2.8.  Statistical analysis . 53  3.3.  Results 54  3.3.1.  Growth inhibitory effects of methoxystilbenes on Hepa1c1c7 cells 54  3.3.2.  Induction of NQO1 activity by methoxystilbenes 57  3.3.3.  Radical quenching activity of methoxystilbenes 60  3.4.  Discussion 61  3.5.  Conclusion 63  Chapter Induction of NQO1 Activity by Indolinones and Related Compounds . 64  4.1.  Introduction 64  4.2.  Experimental Methods . 64  4.2.1.  Materials and cell lines . 64  4.2.2.  MTT assay for determination of cell viability 64  4.2.3.  Determination of NQO1 activity 64  III 4.2.4.  Measurement of 7-ethoxyresorufin O-deethylase (EROD) activity in Hepa1c1c7 cells 64  4.2.5.  Statistical analysis . 65  4.3.  Results 65  4.3.1. Induction of NQO1 activity in Hepa1c1c7 cells 65  4.3.2. Induction of NQO1 activity in the mutant Hepa1c1c7 cell line (c1) . 72  4.3.3.  Induction of CYP1A1 activity by indolinones and related compounds . 79  4.4.  Discussion 87  4.5.  Conclusion 91  Chapter Structure Activity Relationships of NQO1 Induction by Indolinones and Related Compounds 92  5.1  Introduction 92  5.2  Experimental Methods . 93  5.3  Results and Discussion . 94  5.4  Conclusion and Summary 103  Chapter Antiproliferative Activities of Methoxystilbenes and Class 1-4 Indolinones and Related Compounds . 105  6.1.  Introduction 105  6.2.  Experimental Methods . 105  6.2.1.  Determination of antiproliferative activity by the microculture tetrazolium (MTT) assay . 105  6.2.2.  Determination of the effects of test compounds (5-7, 9,10, 37, 42, 45-48) on the cell cycle of HCT116 cells by cell cytometry 106  6.3.  Results 107  IV 6.3.1.  Antiproliferative activity of methoxystilbenes on MCF7, HCT116 and CCL186 cell lines . 107  6.3.2.  Antiproliferative activity of indolinones on MCF7, HCT116 and CCL186 cell lines 110  6.3.3.  Effect of selected class and compounds on cell cycle of HCT116 cells…. ……………………………………………………………………113  6.4.  Discussion 123  6.5.  Conclusion and Summary 126  Chapter Conclusions and Future Work 127  References 131  Appendix 145  V Publications and Conferences 1. Zhang, W.; Go, M. L., Quinone reductase induction activity of methoxylated analogues of resveratrol. Eur J Med Chem 2007, 42 (6), 841-50. 2. Zhang, W.; Go, M. L., Functionalized 3-Benzylidene-indolin-2-ones: Inducers of NAD(P)H: Quinone Oxidoreductase (NQO1) with antiproliferative activity, Bioorg. Med. Chem. 2009, doi:10.1016/j.bmc.2008.12.052 3. Go ML，Zhang W, Functionalized Indolinones，United States Patent，pending US Provisional Patent Application No.61/105,206， pp 26, 2008. 4. Zhang, W.; Go, M. L., Functionalized 3-Benzylidene-indolin-2-ones and related compounds as Inducers of NAD(P)H: Quinone Oxidoreductase (NQO1) in Hepa1c1c7 cells: Structure Activity Relationships, submitted 2008. 5. Zhang Wei, Loh Teck Peng, Mei Lin Go, Antioxidant activity of methoxylated stilbenes related to resveratrol, 17th Singapore pharmacy congress 1-3 July 2005, Singapore. 6. Zhang Wei and Mei Lin Go, Methoxylated stilbenes as inducers of NAD(P)H: Quinone reductase type 1, XIXth International Symposium on Medicinal Chemistry, 29 August-2 September 2006, Istanbul, Turkey. 7. Zhang Wei and Mei Lin Go, Oxygenated Stilbenes as Chemopreventive Agents: Quinone Reductase Induction and Antioxidant Properties, 9th International Symposium by Chinese Organic Chemists & 6th International Symposium by Chinese Inorganic Chemists, 17-20 Dec 2006, Singapore. VI Summary The aim of this thesis was to test the hypothesis that structural modifications of the stilbene resveratrol, a known chemopreventive agent, would result in compounds with greater phase II enzyme induction activity. To this end, two series were designed, synthesized and characterized. The first series comprised of 23 methoxystilbenes. Methoxy groups were introduced in the hope that they would deter the rapid metabolic degradation associated with phenolic hydroxyl groups. Methoxystilbenes had also been associated with strong antiproliferative activities that could contribute to the overall chemopreventive profile of the compound. The second series was based on the replacement of the phenolic ring B of resveratrol with a bioisosteric indolin-2-one moiety. Sixty one compounds were synthesized and they were organized into classes: Classes and 2: 3-substituted indolin-2-ones; Class 3: miscellaneous compounds related to 3-substituted indolin-2-ones; Class 4: isoindigos. Both libraries were evaluated for induction of NAD(P)H: quinone oxidoreductase (NQO1) on the mouse hepatoma Hepa1c1c7 cells. Selected members were evaluated on a mutant cell line (c1) that lacked a functional CYP1A1 gene. They were also evaluated for induction of CYP1A1 activity on Hepa1c1c7 cells using the EROD assay. The antiproliferative activities of the compounds were investigated on human breast cancer cell (MCF7) and colon cancer (HCT116) cell lines, as well as a normal cell line (CCL186). Improved NQO1 induction activity compared to resveratrol was found in some methoxystilbenes. Good activity was associated with the E isomer and the absence of hydroxyl groups on ring A. The most promising compound S3E (E-2,4'dimethoxystilbene) had a CD (concentration required to increase basal NQO1 activity by two fold in Hepa1c1c7 cells) of 0.85 M. S3E had a bifunctional induction profile VII because it also induced CYP1A1 activity. However, it induced NQO1 activity to a greater extent. An active metabolite generated by CYP1A1 might be involved in its induction activity. Antiproliferative activity was found mainly among the Z- methoxystilbenes, in particular those with methoxy groups on positions and of ring A. The different structural requirements for NQO1 induction and antiproliferative activities implied that it would be difficult to combine both properties in the same molecule. The indolinones and isoindigos of the second series yielded several potent NQO1 inducers, with CD values in the nanomolar range. The variation in induction activity in this library was more than 104 fold and this permitted useful SAR to be proposed: These were (i) retention of the nitrogen-linked Michael acceptor moiety in the indolinone template, (ii) the presence of electron withdrawing groups on ring B for the monosubstituted Class compounds, (iii) the relative importance of substitution on ring B compared to ring A substitution in Class compounds, and (iv) the presence of at least two electron withdrawing groups on the isoindigo ring system. Like the methoxystilbenes, the indolinones and isoindigos were found to be bifunctional inducers of NQO1, with the possible involvement of an active metabolite in the induction process. An important observation was the correlation between potent NQO1 induction and the lack of preferential induction of NQO1 compared to CYP1A1. Thus, the isoindigos of Class which were the most potent NQO1 inducers in this series induced NQO1 and CYP1A1 activities to almost the same extent. On the other hand, compounds that were moderately active NQO1 inducers (CD 0.1-0.25 M) induced NQO1 to a greater degree than CYP1A1. A QSAR analysis of the NQO1 induction activity was carried out using principal component analysis (PCA) and projection to latent structures by partial least VIII 10. Tron, G. C.; Pagliai, F.; Del Grosso, E.; Genazzani, A. A.; Sorba, G., Synthesis and cytotoxic evaluation of combretafurazans. J Med Chem 2005, 48, (9), 3260-8. 11. Murias, M.; Handler, N.; Erker, T.; Pleban, K.; Ecker, G.; Saiko, P.; Szekeres, T.; Jager, W., Resveratrol analogues as selective cyclooxygenase-2 inhibitors: synthesis and structure-activity relationship. Bioorg Med Chem 2004, 12, (21), 5571-8. 12. Mylona, A.; Nikokavouras, J.; Takakis, I. M., Preparation of (E)-2-hydroxy-4'substituted stilbenes. J. Chem. Res., Synop. 1986, (12), 433. 13. Lion, C. J.; Matthews, C. S.; Stevens, M. F.; Westwell, A. D., Synthesis, antitumor evaluation, and apoptosis-inducing activity of hydroxylated (E)stilbenes. J Med Chem 2005, 48, (4), 1292-5. 14. Joseph, B.; Darro, F.; Behard, A.; Lesur, B.; Collignon, F.; Decaestecker, C.; Frydman, A.; Guillaumet, G.; Kiss, R., 3-Aryl-2-quinolone derivatives: synthesis and characterization of in vitro and in vivo antitumor effects with emphasis on a new therapeutical target connected with cell migration. J Med Chem 2002, 45, (12), 2543-55. 15. Solladie, G.; Pasturel-Jacope, Y.; Maignan, J., A re-investigation of resveratrol synthesis by Perkins reaction. Application to the synthesis of aryl cinnamic acids. Tetrahedron 2003, 59, (18), 3315-3321. 16. Potter, G. A.; Patterson, L. H.; Burke, M. D.; Butler, P. C. Preparation of diarylethenes and analogs as antitumor prodrugs. 99-GB155 9940056, 19990202., 1999. 17. Perregaard, J.; Thomsen, I.; Lawesson, S. O., Organophosphorus compounds. XI. Oxidation of aromatic compounds with sulfur in hexamethylphosphoric triamide 174 (HMPA). New method for preparation of N,N-dimethylthiocarboxamides. Acta Chem. Scand., Ser. B 1975, 29, (5), 538-44. 18. Corsico Coda, A.; Gamba Invernizzi, A.; Righetti, P. P.; Tacconi, G.; Gatti, G., (Z)- and (E)-Arylidene-1,3-dihydroindol-2-ones: configuration, conformation, and infrared carbonyl stretching frequencies. J. Chem. Soc., Perkin Trans. 1984, (4), 615-19. 19. Blake, K. W.; Jaques, B., Anisotropic effects in alpha -substituted methoxystilbenes. J. Chem. Soc., Perkin Trans. 1973, (12), 1660-3. 20. Bouerat, L. M. E.; Fensholdt, J.; Nielsen, S. F.; Liang, X.; Havez, S. E.; Andersson, E. C.; Jensen, L.; Hansen, J. R. Preparation of indolinone derivatives as agents to prevent, treat and/or ameliorate multiple sclerosis. 2004-DK875 2005058309, 20041216., 2005. 21. Andreani, A.; Rambaldi, M.; Locatelli, A.; Bossa, R.; Galatulas, I.; Ninci, M., Synthesis and cardiotonic activity of 2-indolinones. Eur. J. Med. Chem. 1990, 25, (2), 187-90. 22. Andreani, A.; Burnelli, S.; Granaiola, M.; Leoni, A.; Locatelli, A.; Morigi, R.; Rambaldi, M.; Varoli, L.; Kunkel, M. W., Antitumor activity of substituted E-3(3,4,5-trimethoxybenzylidene)-1,3-dihydroindol-2-ones1. J Med Chem 2006, 49, (23), 6922-4. 23. Olgen, S.; Akaho, E.; Nebioglu, D., Synthesis and anti-tyrosine kinase activity of 3-(substituted-benzylidene)-1, 3-dihydro-indolin derivatives: investigation of their role against p60c-Src receptor tyrosine kinase with the application of receptor docking studies. Farmaco 2005, 60, (6-7), 497-506. 175 24. Andreani, A.; Rambaldi, M.; Locatelli, A.; Bongini, A.; Bossa, R.; Galatulas, I.; Ninci, M., Synthesis and cardiotonic activity of pyridylmethylene-2-indolinones. Eur. J. Med. Chem. 1992, 27, (2), 167-70. 25. Bouerat, L.; Fensholdt, J.; Liang, X.; Havez, S.; Nielsen, S. F.; Hansen, J. R.; Bolvig, S.; Andersson, C., Indolin-2-ones with high in vivo efficacy in a model for multiple sclerosis. J Med Chem 2005, 48, (17), 5412-4. 26. Pandit, B.; Sun, Y.; Chen, P.; Sackett, D. L.; Hu, Z.; Rich, W.; Li, C.; Lewis, A.; Schaefer, K.; Li, P. K., Structure-activity-relationship studies of conformationally restricted analogs of combretastatin A-4 derived from SU5416. Bioorg Med Chem 2006, 14, (19), 6492-501. 27. Volk, B.; Simig, G., New one-pot synthesis of 3-alkyl- and 3-(whydroxyalkyl)oxindoles from isatins. Eur. J. Org. Chem. 2003, (20), 3991-3996. 28. Hirao, K.; Morii, N.; John, T.; Takahashi, S., Rhodium-catalyzed carbonylation of 2-alkynylanilines: syntheses of 1,3-dihydroindol-2-ones. Tetrahedron Lett. 1995, 36, (35), 6243-6. 29. Burger, U.; Bringhen, A. O., Cyclization studies with N-Mannich bases of 2- substituted indoles. Helv. Chim. Acta 1989, 72, (1), 93-100. 30. Leclerc, S.; Garnier, M.; Hoessel, R.; Marko, D.; Bibb, J. A.; Snyder, G. L.; Greengard, P.; Biernat, J.; Wu, Y. Z.; Mandelkow, E. M.; Eisenbrand, G.; Meijer, L., Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors? J Biol Chem 2001, 276, (1), 251-60. 31. Wahl, A.; Fericean, G., The chloroisoindigotins. Ann. Chim. Appl. 1928, 9, 277315. 176 Appendix 3: X-ray crystal data for 21 and 37 X-ray crystal data for 21 Table 1. Crystal data and structure refinement for 7025. Identification code 7025 Empirical formula C17 H15 N O Formula weight 249.30 Temperature 223(2) K Wavelength 0.71073 Å Crystal system Monoclinic Space group P2(1)/n Unit cell dimensions a = 12.440(2) Å b = 4.9770(10) Å 　= 90°. 　= 105.446(4)°. Volume Z Density (calculated) Absorption coefficient F(000) Crystal size Theta range for data collection Index ranges Reflections collected Independent reflections Completeness to theta = 25.00° Absorption correction Max. and min. transmission Refinement method Data / restraints / parameters Goodness-of-fit on F2 Final R indices [I>2sigma(I)] R indices (all data) Largest diff. peak and hole c = 22.227(4) Å 　 = 90°. 1326.5(4) Å 1.248 Mg/m3 0.077 mm-1 528 0.40 x 0.14 x 0.10 mm3 1.71 to 25.00°. -14[...]... group Most of the bioisosteres in Figure 1-4 have the advantage of retaining both H bond donor and acceptor properties, and at same time, circumventing the metabolic susceptibility of the OH group Of the various bioisosteres available, the indolin-2-one was chosen for the present work because of the synthetic accessibility of this moiety from commercial sources which would facilitate synthesis There are... revisit the effect of structurally modifying the stilbenoid framework with the aim of discovering more potent inducers of chemoprotective phase II enzymes 1.7 Statement of Purpose The preceding section has highlighted the remarkable biological profile of resveratrol as well as some of its limitations These are the pleiotropic nature of resveratrol coupled with modest potencies for several activities, and. .. captured the constitutive, molecular orbital related, geometric, lipophilic and electronic properties of the compounds were used in the analysis This analysis provided two important findings, namely that (i) the isoindigos differed from the 3-substituted indolin-2-ones and other compounds in terms of the following descriptors: the number of H bond donors, VDW surface area of these donors and LUMO energies;... the benzyl bromide which was converted to the phosphonium salt by reaction with triphenylphosphine The phenyl ring of the phosphonium salt (S2, Scheme 2-2) would eventually be ring B of the final compounds (S3-S14) In the presence of butyllithium as base, the phosphonium salt S2 was converted to the ylid, which reacted with the substituted benzaldehyde to give the stilbene Both Z and E isomers of the. .. of 20 configuration It may be of interest to note that stilbene S11E was synthesized without the need to protect the 2-OH group on ring A, possibly because of the ortho position of this group The same procedure was adopted for the synthesis of stilbene S14 but the silyloxystilbene was obtained as a mixture of E and Z isomers These were separated by column chromatography and each isomer was separately... to the close proximity of the XRE and ARE in the regulatory region of NQO1 (38) Coordinate induction of the two pathways, if confirmed in future studies, would serve to attenuate the toxic effects of reactive intermediates produced via CYP450 metabolism Most investigators employ the murine hepatoma cells Hepa1c1c7 and its mutant cell lines to investigate the monofunctional /bifunctional character of. .. moiety, and it would be of interest to see if bi- or monofunctional induction would prevail in these compounds To verify these hypotheses, the following investigations will be carried out: (i) Synthesis of a representative library of 3-subsituted indolin-2-ones to determine NQO1 induction activity; (ii) Investigation on the nature of the induction (mono- or bi-functional); (iii) Investigation on the antiproliferative... design of the two classes of target compounds that were investigated for chemopreventive activity These are methoxystilbenes related to resveratrol and the 3-substituted indolin-2-ones which are bioisosteres of phenol Each class is discussed separately, starting from the rationale underlying the design, chemical considerations of the synthetic routes and general experimental methods employed in the syntheses... capacity Thus, their activities may be induced and their induction can occur selectively, without concurrently affecting the activity of phase I enzymes The selective induction of phase II enzymes offers a promising strategy for reducing the risk of carcinogenesis and is considered to be one of the true hallmarks of chemoprevention (18) The induction of the phase II enzyme NAD(P)H: quinone oxidoreductase... desirable monofunctional inducer profile that can be retained even on structural modification Two structural modifications of resveratrol are proposed in this thesis The first modification involves the replacement of the phenolic OH of resveratrol on ring B with 4'-methoxy and the concurrent introduction of hydroxyl or methoxy groups on ring A The presence of the methoxy group would deter the rapid metabolic . NATIONAL UNIVERSITY OF SINGAPORE 2008 INVESTIGATIONS INTO THE CHEMOPREVENTIVE POTENTIAL OF STILBENES, INDOLINONES AND ISOINDIGOS: SYNTHESIS AND MODE OF ACTION STUDIES ZHANG WEI. INVESTIGATIONS INTO THE CHEMOPREVENTIVE POTENTIAL OF STILBENES, INDOLINONES AND ISOINDIGOS: SYNTHESIS AND MODE OF ACTION STUDIES ZHANG WEI (B.Sc.,. (i) the isoindigos differed from the 3-substituted indolin-2-ones and other compounds in terms of the following descriptors: the number of H bond donors, VDW surface area of these donors and