Taking Artemisinin to Clinical Anticancer Applications: Design, Synthesis and Characterization of pH-responsive Artemisinin Dimer Derivatives in Lipid Nanoparticles Yitong Zhang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2015 Reading Committee: Tomikazu Sasaki, Chair Rodney J.Y. Ho Champak Chatterjee Program Authorized to Offer Degree: Chemistry i ©Copyright 2015 Yitong Zhang ii University of Washington Abstract Taking Artemisinin to Clinical Anticancer Applications: Design, Synthesis and Characterization of pH-responsive Artemisinin Dimer Derivatives in Lipid Nanoparticles Yitong Zhang Chair of the Supervisory Committee: Professor Tomikazu Sasaki Chemistry iii Abstract Qinghaosu or Artemisinin is an active sesquiterpene lactone isolated from Artemisia annua L. The natural product and its derivatives are known as a first line treatment for malaria. Investigations have also reported that the compound exhibits anti-cancer activities both on cell lines and in animal models. The remarkably stable endoperoxide bridge under ambient conditions is believed to be responsible for the selectivity as well as potency against cells that are rich in iron content. Dimeric derivatives where two artemisinin units are covalently bonded through lactone carbon (C10) show superior efficacies against both malaria parasites and cancer cells. Artemisinin dimer succinate derivative demonstrates a 100-fold enhancement in potency, compared to the natural product, with IC50 values in the low micromolar range. This work focuses on the development of artemisinin dimer derivatives to facilitate their clinical development. Novel pH-responsive artemisinin dimers were synthesized to enhance the aqueous solubility of the pharmacophore motif. Compounds with promising potency against human breast cancer cell lines were selected for lipid and protein based nanoparticle formulations for delivery of the derivatives without the need of organic co-solvents into animal models. To improve the efficiency in designing appropriate lipid-nanoparticles as drug delivery systems, a geometrical flit model based on the drug binding pocket hypothesis was introduced. Finally, to lay a foundation for understanding in vivo behaviors of artemisinin dimer derivatives and their nanoparticle formulations, the first analytical method for preparation of biological tissue samples and quantification of artemisinin dimer derivative using liquid chromatography-tandem mass spectrometry was developed. iv Table of Contents CHAPTER 1 INTRODUCTION...................................................................................................................................... 4 Reference .................................................................................................................................................................... 11 CHAPTER 2 SYNTHESIS OF PH-RESPONSIVE ARTEMISININ DERIVATIVES THAT INHIBIT GROWTH OF BREAST CANCER CELLS IN VITRO AND INDUCE DOWN- REGULATION OF HER FAMILY MEMBERS .......................................................................... 15 Abstract ...................................................................................................................................................................... 16 Introduction ............................................................................................................................................................... 17 Results ......................................................................................................................................................................... 20 Discussions.................................................................................................................................................................. 28 Experimental Section ................................................................................................................................................ 31 Reference .................................................................................................................................................................... 42 CHAPTER 3 PH-RESPONSIVE ARTEMISININ DIMER IN LIPID NANOPARTICLES ARE EFFECTIVE AGAINST HUMAN BREAST CANCER IN A XENOGRAFT MODEL ............................................ 47 Abstract ...................................................................................................................................................................... 47 Introduction ............................................................................................................................................................... 48 Results and Discussion .............................................................................................................................................. 50 Experimental Section ................................................................................................................................................ 64 Reference .................................................................................................................................................................... 72 1 CHAPTER 4 OPTIMIZATION OF ADP109-LIPID NANOPARTICLES FOR IMPROVED DRUG RETENTION IN VITRO ..................................................................................................................... 74 Abstract ...................................................................................................................................................................... 75 Introduction ............................................................................................................................................................... 76 Results and Discussion .............................................................................................................................................. 79 Experimental Section ................................................................................................................................................ 87 Reference .................................................................................................................................................................... 91 CHAPTER 5 DIFFERENCES IN FATTY-ACYL CHAIN LENGTHS MODULATE THE STABILITY OF HYDROPHOBIC COMPOUND INTEGRATION INTO LIPID BILAYERS: A GEOMETRICAL FIT MODEL FOR PHOSPHOLIPID NANOPARTICLE DESIGN ................................................... 92 Abstract ...................................................................................................................................................................... 93 Introduction ............................................................................................................................................................... 94 Results ......................................................................................................................................................................... 98 Discussions................................................................................................................................................................ 109 Experimental Section .............................................................................................................................................. 113 Reference .................................................................................................................................................................. 118 CHAPTER 6 SAMPLE PREPARATION AND LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY ANALYSIS OF ARTEMISININ DIMER LIPOSOME FROM BIOLOGICAL TISSUES ................................................................................................................................................ 120 Abstract .................................................................................................................................................................... 121 Introduction ............................................................................................................................................................. 122 Results ....................................................................................................................................................................... 124 Discussion ................................................................................................................................................................. 131 2 Experimental Section .............................................................................................................................................. 133 Reference .................................................................................................................................................................. 138 CHAPTER 7 SYNTHESIS OF PH-RESPONSIVE ARTEMISININ DIMER BODIPY DERIVATIVES; BOVINE SERUM ALBUMIN-BOUND PARTICLE PREPARATION AND EVALUATION OF EFFICACY ON XENOGRAFT MODEL ................................................................................................................. 140 Abstract .................................................................................................................................................................... 141 Introduction ............................................................................................................................................................