Olga Genilloud obtained her Ph.D. in Chemistry (Biochemistry and Molecular Genetics) in 1988 from the Universidad Complutense de Madrid, Spain. After a short stay at the Microbiology Department at Harvard Medical School in Boston, in 1989 she joined the Centro de Investigacion Basica de Espana (CIBE), at Merck Sharp and Dohme in Spain, where she has led for nineteen years the discovery of bacterial natural products as new leads for drug development. In February 2009 she was appointed Scientific Director of Fundacion MEDINA. She is a member of many different scientific societies and has extensive experience in the coordination and management of international scientific working groups and is actively contributing to different Master and Pre-doctoral teaching programs. M¬ Francisca Vicente has a Ph.D. in Biology and Molecular Genetics (1986, Universidad Autonoma de Madrid) and more than 10 years experience in R&D, managing teams and working in the multidisciplinary environment of multinational pharmacy (Merck Sharp and Dohme, Spain) and other research centres (Institute Pasteur, and Hospital Necker, France). She has been in charge of the design, implementation, and supervision of many R&D projects. She is experienced in identifying and managing the economic resources for developing new scientific and technological projects. She has coordinated and managed national and international research groups. Her teaching experience was gained by giving practical and theoretical classes in different masters and university programs. In February 2009, she joined the Fundacion MEDINA as Area Head of the Screening and Target Validation Department.

Drug Discovery from Natural Products

By Olga Genilloud, Francisca Vicente

The Royal Society of Chemistry

Copyright © 2012 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-361-8

Contents

Section 1 New Approaches to Exploit Natural Products Chemistry,
1.1 Semisynthesis/Synthesis de novo of Natural Products to Cope with Supply Issues,
Chapter 1 Semisynthesis Approach of Ecteinascidin 743 (ET-743, Yondelis®) Carmen Cuevas and Andrés Francesch, 5,
Chapter 2 Chondramides and Chivosazoles – Two Metabolites Which Interfere with the Actin Cytoskeleton Lynette A. Smyth, Tobias Brodmann and Markus Kalesse, 17,
Chapter 3 Class III Lantibiotics – an Emerging Family of Thioether-Containing Peptides Bartlomiej Krawczyk, Joanna M. Krawczyk and Roderich D. Süssmuth, 42,
Chapter 4 Mutant Manufacturers Andreas Kirschning, Simone Eichner, Jekatherina Hermane and Tobias Knobloch, 58,
Chapter 5 Progress in Enhancing the Neurotrophic Effects of Natural FKBP Ligands Guy T. Carter, 79,
1.2 Engineering Natural Product Synthetic Pathways and Genome Mining,
Chapter 6 Biosynthesis of Indolocarbazole Alkaloids and Generation of Novel Derivatives by Combinatorial Biosynthesis Carmen Méndez, Francisco Moris and José A. Salas, 99,
Chapter 7 New Lantibiotics from Natural and Engineered Strains Sonia I. Maffioli, Paolo Monciardini, Margherita Sosio and Stefano Donadio, 116,
Chapter 8 Mining Microbial Genomes for Metabolic Products of Cryptic Pathways D. Oves-Costales and G. L. Challis, 140,
Chapter 9 Mining Cyanobacterial Genomes for Drug-Like and Bioactive Natural Products John A. Kalaitzis and Brett A. Neilan, 159,
Chapter 10 Epigenetic Approaches to Natural Product Synthesis in Fungi Alexandra A. Soukup and Nancy P. Keller, 198,
Section 2 New Methodologies and Screening Technologies for the Exploitation of Microbial Resources,
Chapter 11 Novel Approaches to Exploit Natural Products from Microbial Resources Olga Genilloud and Francisca Vicente, 221,
Chapter 12 Discovery and Development of Platensimycin and Platencin Sheo Singh, 249,
Chapter 13 Coupling Chemical Genomics and Natural Products: The Discovery of Parnafungins and Novel Antifungal Leads Hao Wang, Craig A. Parish, Deming Xu and Terry Roemer, 278,
Section 3 Novel Microbial Natural Products and Derivatives,
Chapter 14 Natural Products: New Agents against MDR Tuberculosis Ujjini Manjunatha, Fumiaki Yokokawa, Meera Gurumurthy and Thomas Dick, 307,
Chapter 15 Retapamulin: a First-in-Class Pleuromutilin Antibiotic Rodger Novak, 326,
Chapter 16 Epothilones as Lead Structures for New Anticancer Drugs Bernhard Pfeiffer, Fabienne Zdenka Gaugaz, Raphael Schiess and Karl-Heinz Altmann, 339,
Chapter 17 The Contribution of Marine Chemistry in the Field of Antimalarial Research Ernesto Fattorusso and Orazio Taglialatela-Scafati, 374,
Future Challenges, 391,
Subject Index, 394,

CHAPTER 1

Semisynthesis Approach of Ecteinascidin 743 (ET-743, Yondelis®

CARMEN CUEVAS AND ANDRÉ S FRANCESCH

PharmaMar, S. A., Avenida de los Reyes, 1, Colmenar Viejo, Madrid, 28770, Spain

1.1 Introduction

For thousands of years natural products obtained from terrestrial sources have played a very important role in health care and prevention of diseases. However, it was not until the nineteenth century (1804) that scientists (Friedrich Sertürner) isolated active components (morphine) from various medicinal plants (Papaver somniferum) and since then terrestrial natural products have been extensively screened for their medicinal purposes. Between the years 1981 and 2006, about a 100 anticancer agents have been developed, of which, 25 are natural product derivatives, 18 are natural product mimics, 11 candidates are derived from a natural product pharmacophore, and 9 are pure natural products. In recent years, the chemistry of natural products derived from marine organisms has become the focus of a much greater research effort. This is due in large part to the increased recognition of marine organisms as a source for bioactive compounds with pharmaceutical applications or other economically useful properties. Because of the physical and chemical conditions in the marine environment, almost every class of marine organism possesses the capacity to produce a variety of molecules with unique structural features. These molecules offer an unmatched chemical diversity and structural complexity, together with a biological potency and selectivity. The fact that marine resources are still largely unexplored has inspired scientists from academia and the pharmaceutical industry to intensify their efforts by using novel technologies to overcome the inherent problems in discovering compounds which may have potential for further development as pharmaceuticals or as functional products such as cosmetics, nutritional supplements, and functional foods. These efforts have resulted in the development of around 15 marine natural products in various phases of clinical development, mainly in the oncology area, that includes the PharmaMar compounds: Yondelis®, Aplidin®, Irvalec®, Zalypsis®, PM01183, and PM060184.

1.2 Isolation of Ecteinascidin 743

Ecteinascidia turbinata Herdman (1880) (family Perophoridae) is a colonial ascidian (tunicate) species from the Caribbean and the Mediterranean that belongs to the class Ascidiacea within the subphylum Tunicata (also called Urochordata) possessing a transparent, orange or whitish-colored tunic. Ascidians, or sea squirts, are small, bottom-dwelling soft-bodied marine animals that form colonies comprising many individuals, called zooids. The name ‘tunicate’ is derived from their characteristic protective covering, or tunic, which functions to a certain extent as an external skeleton and consists of some cells, blood vessels, and a secretion of a variety of proteins and carbohydrates, including tunicin, a cellulose-like polymer – an unusual finding in animals. Within the tunic is the muscular body wall, which controls the opening of the siphons used for feeding. A typical colony consists of a dense cluster of elongated, somewhat club-shaped, zooids connected at their bases by a network of stolons that adheres the colony to the surface of the substrate on which it grows. The tunicate normally lives in coastal shallow waters (0 to 15 m depth) and in lagoons growing on red mangroves roots, rocks, shells, sand, and marine meadows. It is distributed throughout the Caribbean and in the temperate regions of the Atlantic and the Mediterranean. Reproduction is through a sexual cycle in which eggs are fertilized, hatched, and brooded internally and the larvae released to the sea as they reach maturity. Asexual reproduction is by budding of new zooids from the base of an existing zooid or from the stolon mass of the colony. Whereas dispersion of the species is facilitated as a consequence of the larvae being carried to new locales by ocean currents, the role of the stolon constitutes an important adaptive strategy for regeneration and growth, assuring fast colonization and extension of extant colonies over available surfaces of both natural and artificial substrata.

Aqueous ethanol extracts of Ecteinascidia turbinata were shown to have antitumor effects in 1969, but isolation and structural characterization of the active compounds was not achieved until 1990 when Rinehart and co-workers reported six new chemical entities called ecteinascidins (ETs), 743 (1), 729 (2), 745 (3), 759A (4), 759B (5), and 770 (6), of which ET-743 was the most abundant representative (0.0001% yield). Simultaneously, Wright and co-workers described ET-743 and 729. The novel and unique chemical structures of ecteinascidins, determined by extensive NMR and mass spectral studies, is formed by a monobridged pentacyclic skeleton composed of two fused tetrahydroisoquinoline rings (subunits A and B) linked to a 10-membered lactone bridge through a benzylic sulfide linkage. Most ecteinascidins have an additional tetrahydroisoquinoline or tetrahydro-β-carboline ring (subunit C) attached to the rest of the structure through a spiro ring (Figure 1.1). This is one of the features distinguishing these molecules from saframycins, safracins, and renieramycins, compounds isolated from bacterial sources and sponges.

1.3 Mechanism of Action

In contrast to traditional alkylating agents that bind guanine at the N7 or O6 position in the DNA major groove, ET-743 is the first of a new class of DNA binding agents with a complex, transcription-targeted mechanism of action. ET743 binds the exocyclic N2 amino group of guanines in the minor groove of DNA with preference for GC-rich triplets through an iminium intermediate generated in situ by dehydration of the carbinolamine moiety present in the monobridge pentacycle skeleton. The binding of ET-743 in the minor groove induces the formation of DNA adducts, which bend DNA towards the major groove. The resulting covalent adduct is additionally stabilized through van der Waals interactions and one or more hydrogen bonds between the monobridge pentacycle skeleton with neighboring nucleotides in the same or opposite strand of the DNA double helix, thus creating the equivalent to a functional interstrand crosslink.

The additional subunit C apparently does not participate in DNA binding and it was proposed to protrude out of the DNA, being able to interact with different DNA-binding proteins located in the DNA adduct area. One of these proteins is XPG endonuclease, a member of the nucleotide excision repair (NER) system. Moreover, ET-743 is apparently blocking the trans-activating ability of chimeric proteins such as FUS-CHOP or EWS1-Fli1 modulating the transcription of genes that should be crucial for tumorigenesis in specific cancer subtypes. At the cell cycle level, these events result in a decrease in the rate of progression of the tumor cells through the S phase toward G2 or as a prolonged G2-phase blockade.

1.4 Preclinical Drug Development

Preclinical data generated during the development of ET-743 have provided important insight for the selection and design of the clinical trials. Early in vitro studies carried out by PharmaMar and the National Cancer Institute (NCI) in a panel of 60 human tumor cells identified the potent activity (1 pM to 10 nM) of ET-743. The NCI COMPARE analysis with more than 100 standard anticancer agents was negative, indicating a new mechanism of action of ET-743.

ET-743 has been tested in a great variety and number of models against tumors of murine origin (P388 leukemia and B16 melanoma), human sensitive xenografts (melanoma, MEXF 989; non-small-cell lung cancer, LXFL 529; breast, MX-1 early and advanced; and ovarian, HOC 22), and human resistant xenografts (melanoma, MEXF 514; non-small-cell lung cancer, LXFL 629; and ovarian, HOC 18). These first efforts showed that ET-743 has a broad spectrum of antineoplastic activity, with several tumor types showing selectivity; namely, melanoma, non-small-cell lung cancer, and ovarian carcinomas. As a further example of strong activity and long-lasting antitumor effects, the action of ET-743 on human endometrial carcinoma xenografts (HEC-1-B) results in complete regression lasting for more than 125 days. The determination that soft tissue sarcomas (STS) are more sensitive to ET-743 than other solid tumors was not predicted during the preclinical development of the drug. The finding was serendipitous and came from the prevalence of responding or stable STS patients in the clinical trials. As a result, there has been a considerable effort to confirm this finding to supplement the extensive nonclinical profile already characterized for the antineoplastic effect of ET743 against other solid tumors. In fact, a variety of sarcomas are differentially sensitive to ET-743, showing IC50 potencies in the picomolar and sub-picomolar range compared to the nanomolar concentrations established against non-STS solid tumors.

1.5 Clinical Studies

Yondelis® (Trabectedin, ET-743) has been designated an orphan drug by the European Commission (EC) and the USA Food and Drug Administration (FDA) for the indications of soft tissue sarcoma (STS) and ovarian cancer.

Soft tissue sarcomas are malignant tumors that originate in the soft tissues of the body. Soft tissues connect, support, and surround other body structures. The soft tissues include muscle, fat, blood vessels, nerves, tendons, and synovial tissues. The annual incidence of STS in Europe is approximately 0.004% (4 in 100 000 people). Five-year overall survival (OS) rates are on the order of 50 to 60%, irrespective of disease stage at diagnosis. Within 2 to 3 years from diagnosis, approximately 30 to 50% of patients develop metastases despite optimal treatment for localized disease.

Ovarian cancer is one of the deadliest gynecological cancers. Unfortunately, detection of ovarian cancer is difficult, and the disease is often diagnosed too late for successful treatment. Although with the current standard of care (debulking surgery followed by platinum-based chemotherapy) most of the patients achieve a complete clinical remission, eventually the majority of them will relapse and die owing to their disease.

In September 2007, Yondelis® received marketing authorization from the European Commission for the treatment of patients with advanced or metastatic soft tissue sarcoma after failure of anthracyclines and ifosfamide, or who are unsuited to receive these agents. In September 2009 Committee for Medicinal Products for Human Use (CHMP) adopted a positive opinion on a variation to extend the indication for Yondelis® for the treatment of patients with relapsed platinum-sensitive ovarian cancer in combination with pegylated liposomal doxorubicin (PLD).

Worldwide, more than 12 000 patients in more than 63 countries have already been treated with this innovative drug and shown a good safety and tolerability profile. The most frequent adverse event appears to be neutropenia, which is reversible. Transaminase elevations were also reported but were transient. No mucositis, alopecia, neurotoxicity, cardiotoxicity, or cumulative toxicities have been observed.

In other indications, such as breast and prostate cancer, Yondelis® is currently being studied in Phase II clinical trials trying to identify the patients that should respond to the drug treatment by measuring levels of the endonuclease XPG. Yondelis® is also being tested in pediatric indications.

1.6 Chemical Synthesis

Ecteinascidia turbinata has been successfully grown and harvested in aquaculture facilities located along the Mediterranean coast. The purification of the active ingredient was then accomplished on an industrial scale, using chromatographic procedures that represent a more practical and environmentally sound practice than harvesting the creature from the wild. Nevertheless, in recent years several synthetic schemes have been developed for industrial production of ET-743 in the quantities and quality required for a drug product (Yondelis®) that will be used in worldwide clinical studies and manufacturing for commercialization.

1.6.1 Synthetic Routes to Ecteinascidin 743 (ET-743, Yondelis®)

To date, three distinct total synthetic routes to Yondelis® (ET-743) have been reported. The pioneer work of E. J. Corey and co-workers provided for the first time a total synthesis of this complex molecule in 1996. This breakthrough scheme resolved one of the main roadblocks to the synthesis – the cyclization to obtain the 10-membered ring – is based on the elegant maneuver of the generation of the short-lived ortho-quinone methide and attack by cysteine thiol. Five years later, Fukuyama and co-workers published a second total synthesis of ET-743, based in part on previous efforts targeting members of the saframycins and renieramycins, in which the cyclization reaction takes place in the conditions previously developed by Danishefsky and co-workers to converge with the general approach established by Corey for the later stages of the synthesis. Finally in 2006, a highly convergent total synthesis of ET-743 was reported by Zhu and co-workers as a conclusion of previous investigations on the synthesis of this family of compounds. This third total synthesis has been achieved in 31 steps in the longest linear sequence from 3- methyl catechol. Additionally, two formal syntheses have been described by the Danishefsky and Williams groups when describing the synthesis of an advanced intermediate in the Fukuyama total synthesis and an intermediate of the first formal synthesis, respectively.

The procedures outlined above represent some of the most outstanding work in recent synthetic organic chemistry. However, the long and involved procedures for total synthesis of the molecule represent a tremendous barrier to industrial manufacture of the drug, which is particularly challenging in the face of regulatory requirements for pharmaceuticals. This problem was finally solved with the development of a semisynthetic procedure representing the first industrially feasible route to the manufacture of the drug on a large scale. The procedure uses cyanosafracin B (11), an antibiotic available through fermentation of the bacteria Pseudomonas fluorescens, as the starting point. This approach is similar to traditional semisynthetic approaches, though in this case the semisynthetic product is a difficult-to-source natural product. Optimization of the fermentation process, followed by its transformation according to Scheme 1.1, provided a robust, easily scaled-up procedure for manufacturing the drug.

(Continues…)Excerpted from Drug Discovery from Natural Products by Olga Genilloud, Francisca Vicente. Copyright © 2012 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.