SYNTHESES AND BIOLOGICAL ACTIVITY OF SPARSOMYCIN AND ANALOGS THE CHEMISTRY OF CHIRAL FUNCTIONALIZED SULFOXIDES AND SULTINES DERIVED FROM CYSTEINE ROB M.J. LISKAMP SYNTHESES AND BIOLOGICAL ACTIVITY OF SPARSOMYCIN AND ANALOGS THE CHEMISTRY OF CHIRAL FUNCTIONALIZED SULFOXIDES AND SULTINES DERIVED FROM CYSTEINE Promotor : Prof. Dr. R.J.F. Nivard Co-referent : Dr. H.C.J. Ottenheijm SYNTHESES AND BIOLOGICAL ACTIVITY OF SPARSOMYCIN AND ANALOGS THE CHEMISTRY OF CHIRAL FUNCTIONALIZED SULFOXIDES AND SULTINES DERIVED FROM CYSTEINE PROEFSCHRIFT TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE WISKUNDE EN NATUURWETENSCHAPPEN AAN DE KATHOLIEKE UNIVERSITEIT VAN NIJMEGEN OP GEZAG VAN DE RECTOR MAGNIFICUS PROF. DR. J.H.G.I. GIESBERS VOLGENS HET BESLUIT VAN HET COLLEGE VAN DEKANEN IN HET OPENBAAR TE VERDEDIGEN OP VRIJDAG 10 DECEMBER 1982 DES NAMIDDAGS OM 4 UUR DOOR ROBERTUS MATTHIAS JOSEPH LISKAMP GEBOREN TE NIJMEGEN NIJMEGEN 1982 Dit proefschrift vormt de neerslag van vier jaar wetenschappelijk onderzoek. Ik wil iedereen - en dat waren er zeer velen - bedanken, die hieraan gedurende die tijd door hun stimulerende discussie of door de feitelijke uitvoering deel hadden. Zij gaven mede richting en vorm, niet alleen organisch maar ook biochemisch/biologisch en fysisch chemisch, aan het verrichte onderzoek. Daarnaast wil ik hen bedanken die een bijdrage geleverd hebben aan de onmis­ bare technische uitvoering van het in dit proefschrift beschreven onderzoek. En tenslotte wil ik ook hen bedanken die aan de vormgeving van het proef­ schrift hun medewerking hebben verleend. Tekeningen : Wim van Luijn en de afdeling illustratie Type-werk : Dorine Hesselink-Balvert Afbeelding op de omslag: Een 'stacked plot' van een 2D-spin-echo correlated spectrum van synthetisch sparsomycine verzorgd door Wim Guijt This research was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO). Het leven is een pijpkaneel CONTENTS CHAPTER I General intractuation. 1 Introduction to the Chapters. 7 CHAPTER II 9 Absolute Configuration of Sparsomyoin. A Chiroptical Study of Sulfoxides. H.C.J. Ottenheijm, R.M.J. Liskamp, P. Helquist, J.W. Lauher, M.S. Shekhani, J. Am. Chem. Soo. (1981), 103, 1720. CHAPTER III 13 Total Synthesis of the Antibiotic Sparsomycinj a Modified Uracil Amino Add Mono-oxodithioacetal. H.C.J. Ottenheijm, R.M.J. Liskamp, S.P.J.M. van Nispen, H.A. Boots, M.W. Tijhuis, J. Org. Chem. (1981), 46, 3273. CHAPTER IV 24 Synthesis and Ring-Opening Reactions of Functionalized Sultines. A new Approach to Sparsomyein. R.M.J. Liskamp, H.J.M. Zeegers, H.C.J. Ottenheijm, J. Org. Chem. (1981), 46, 5408. CHAPTER V 1. Interactions of sparsomyoin with the peptidyttransferase 30 center of ribosomes 2. Inhibition of the 'protein' synthesis in yeast 38 (Saccharomyces Cerevisae) cell-free systems by sparsomyoin and analogs; preliminary results of a structure-activity relationship study. 3. Structure-Activity Relationships of Sparsomyoin and 40 Analogs; Octylsparsomycin: the First Analog More Active than Sparsomyoin R.M.J. Liskamp, J.H. Colstee, H.C.J. Ottenheijm, P. Lelieveld, W. Akkerman, J. Med. Chem. submitted for publication 4. The antitumor activity of sparsomyoin 53 CHAPTER VI 56 Conformational Analysis of Funotionalized SulbLnes by Nuclear Magnetic Resonance and X-Ray Crystallography ; Application of a Generalized Karplus equation. C.A.G. Haasnoot, R.M.J. Liskamp, P.A.W. van Dael, J.H. Noordik, H.C.J. Ottenheijm, J. Am. Chem. Soc., submitted for publication. CHAPTER VII 76 Flash Vacuum Thermolysis of Funotionalized y-Sultines R.M.J. Liskamp, H.J.M. Blom, R.J.F. Nivard, H.C.J. Ottenheijm, J. Org. Chem., submitted for publication. CHAPTER VIII 84 Approaches to the marasmin fragment of y-glutamylmarasmin SAMENVATTING 97 CURRICULUM VITAE 101 CHAPTER I GENERAL INTRODUCTION INTRODUCTION TO THE CHAPTERS GENERAL INTRODUCTION The isolation of sparsomycin 1^, next to tubericidin (sparsomycin A) 21, from a Streptomyoes Sparsogenes broth was reported by Argoudelis and Herr in 19622'3. More recently this compound has also been obtained from Stvevtomyaes Cuspidosporus1* »5. After its isolation it was roughly characterized (UV, IR, elementary analysis, specific rotation and molecular weight) by Argoudelis and Herr . ОН о "H2 Us. ΐ ON OH 1 Sparsomycin (Sc- Rs) 2 tubericidin Subsequently a first evaluation of its biological activity was carried out by Owen, Dietz and Camiener6. In their experiments sparsomycin was found to be active against KB human epidermoid carcinoma cells in tissue culture. The compound was very active against these mammalian cells (KB cell protein synthesis was inhibited 50% (ID50) at a concentration of 0.05 pg/ml), and moderately active against a variety of gram negative and gram positive bacteria as well as against fungi. In VÌVO data showed inhibition of growth of several tumors as assayed from tumor diameter measurements or from tumor weights. The toxicological screening of sparsomycin indicated an acute LD50 value of 2.4 mg/kg in mice. These data about the biological activity were probably the immediate cause to test sparsomycin in a phase I clinical study in 19647. In this study with 5 patients who had all advanced carcinomas or sarcomas, the drug was daily administered i.V. for an intended period of 42 days. The dose given ranged from 0.085 mgAg - 0.24 mg/kg. Two patients noted difficulty of vision, one after 13 days of treatment (total dose 12 mg) and one after 15 days of treatment (total dose 7.5 mg), where­ after treatment was stopped. It was conceived, that the primary biological activity of sparsomycin was due to a strong inhibition of the protein synthesis rather than inhi­ bition of DNA or RNA synthesis. Its effect on the protein synthesis was demonstrated by the decline of the protein synthesis of intact prokaryotic cells Ч6/8-12, eukaryotic cells13"^0 - including transformed6'0'9'19'20 and/or virus infected cells » ; and in various cell-free systems (pro- karyotes13'23-26'34, eukaryotes27-41). The behavior of sparsomycin with regard to its inhibitory action and influence on the polyribosomes has also been investigated гп vivoi*2-~t*7. There is ample evidence4"»^9 that sparsomycin inhibits the protein synthesis by interacting with the peptidyltransferase center of ribosomes, the devices of the cell, where the protein synthesis actually takes place. The peptidyltransferase center is responsible for the cruxial event in the elongation step of the protein synthesis in which the peptidyl chain of the peptidyl-tRNA is transferred to the aminoacyl-tRNA. 2 The pronounced inhibition of the protein synthesis may be - at least partly - responsible for several reported secondary actions of sparsomycin, such as the inhibition of the RNA-synthesis13, the DNA-synthesis16 and perhaps also for the reported ocular toxicity of sparsomycin6»50. Because sparsomycin is a selective and effective inhibitor of the protein synthesis, it has been used as a tool in a number of other biochemical studies15»51~60. Our interest in sparsomycin was roused by the discrepancy that existed between the wealth of information on the biological activity as well as the biochemical mechanism of action (vide supra) on one hand and the limited knowledge of the organic chemistry of the molecule on the other hand: when we started our research on sparsomycin there was no total synthesis of the molecule available. To our opinion the development of a flexible synthesis or synthetic methodologies for a molecule possessing an interesting biological activity is an absolute prerequisite for thorough studies on the biological activity and/or biochemical mechanisms of interaction. This can also be concluded from the work on - especially - puromycin (Chapter V), on chloramphenicol and on lincomycin, which interfere with the peptidyltransferase center too. As a consequence we started our investigations on sparsomycin, directing our first efforts towards a total synthesis of this molecule. A total synthesis was feasible since the structure of sparsomycin - excepting the chirality of the sulfoxide moiety - had been elucidated by Wiley and MacKellar61 in 1970. Our purpose was to develop a flexible synthesis that would enable us to prepare a wide variety of analogs. It was planned to use these analogs in structure activity relationship studies (see Chapter V) to determine : a^ the structural features and stereochemical requirements, essential for an optimal biological activity b the molecular mechanism of action of sparsomycin and its analogs. In addition, our aim was to study whether structural modifications in sparsomycin might yield a molecule with more selective biochemical and farmacological properties e.g. a molecule that will penetrate preferentially into a transformed cell. Furthermore, our experiences with the syntheses of analogs of sparsomycin will be used to develop a synthesis of a sparsomycin analog, containing a radioactive label as well as an affinity label. The latter may give rise to covalent bond(s) with (a) molecule(s) of its site of interaction. Studies with this affinity analog of sparsomycin might be valuable for an further understanding of ribosome structure and function. The guiding principle in structure-activity relation studies and in affinity label studies has to be the awareness of the existence of a close relation between the structure as well as the chemistry of a molecule and its biological activity. This implies a 'bio-organic chemical' attitude: hypotheses have to be formulated based on results obtained from studies on the biological activity of an effector molecule and on available information about the biochemical/biological structure of the effector's target (e.g. the ribosome). These hypotheses must lead to the design of molecular models. Although the biological activity of sparsomycin was an important reason for directing our synthetic efforts toward this molecule, there were other reasons for embarkment on a total synthesis. First, the development of a total synthesis would allow the determination of the absolute configuration of the sulfoxide-sulfur atom of sparsomycin.