《天然药物化学》课程参考文献（海洋天然产物）Marine Natural Products and Related Compounds in Clinical and Advanced Preclinical Trials.pdf

Marine Natural Products and Related Compounds in Clinical and Advanced Preclinical Trials† David J. Newman* and Gordon M. Cragg Natural Products Branch, Developmental Therapeutics Program, NCI-Frederick, P.O. Box B, Frederick, Maryland 21702 Received February 10, 2004 The marine environment has proven to be a very rich source of extremely potent compounds that have demonstrated significant activities in antitumor, antiinflammatory, analgesia, immunomodulation, allergy, and anti-viral assays. Although the case can and has been made that the nucleosides such as Ara-A and Ara-C are derived from knowledge gained from investigations of bioactive marine nucleosides, no drug directly from marine sources (whether isolated or by total synthesis) has yet made it to the commercial sector in any disease. However, as shown in this review, there are now significant numbers of very interesting molecules that have come from marine sources, or have been synthesized as a result of knowledge gained from a prototypical compound, that are either in or approaching Phase II/III clinical trials in cancer, analgesia, allergy, and cognitive diseases. A substantial number of other potential agents are following in their wake in preclinical trials in these and in other diseases. Introduction The initial discoveries from the marine environment that led to the belief that true marine-derived drugs would not be overly long in reaching the market can be traced to the reports of Bergmann on the discovery and subsequent identification of spongothymidine and spongouridine in the early 1950s from the Caribbean sponge Tethya crypta.1-3 These reports actually led to a complete reversal of the then current dogma, which prior to these discoveries was “that for a nucleoside to have biological activity, it had to have ribose or deoxyribose as the sugar, but that the base could comprise a multiplicity of heterocycles and even carbocycles”. The subsequent explosion of compounds is described with the relevant citations by Suckling4 and Newman et al., 5 and these discoveries led to the identification of a close analogue, cytosine arabinoside, as a potent antileukemic agent; this compound (1) subsequently was commercialized by Upjohn (now Pharmacia) as Ara-C. Other closely related compounds such as adenine arabinoside (Ara-A) (2), an antiviral compound synthesized and commercialized by Burroughs Wellcome (now Glaxo SmithKline) and later found in the Mediterranean gorgonian Eunicella cavolini, and even azidothymidine (AZT) (3) can be traced back to this initial discovery of the “other than ribose-substituted bioactive nucleosides”. The advent of scuba techniques approximately 60 years ago and their subsequent utilization by natural products chemists and biologists working closely with them led to questions such as, Why are certain marine invertebrates not prey for organisms higher up the evolutionary tree? Why do fish not eat particular algae? Why do two sponges grow and expand until they touch, but do not grow over each other? One possibility was that the organisms have some form of chemical communication or defense that enables an individual organism to establish a particular niche and thrive there. One has to realize that these marine invertebrates and marine plants, with very few exceptions, are sessile and require a “foot-hold” on a nonmoving, fixed substrate (rock or coral) that permits them to feed by filtration of the seawater flowing in and around them. Initial attempts at determining the chemistries of marine organisms were simply extensions of tried and true phytochemical techniques. Thus, easily accessible organisms (generally sponges and encrusting organisms such as ascidians) were collected by hand using snorkel or simple scuba systems, and then their chemical components were extracted and identified. Any biological activity was found as an afterthought in these initial experiments (though as shown above, active compounds could be found by these techniques that would ultimately be useful as treatments for human diseases). A corollary to the more systematic searching for marinederived products was that very sensitive analytical tools had to be used, as in general, the amounts of bioactive materials that could be recovered were exceedingly small. There are examples given later in detail, but levels of 1 mg of compound per 3 kg of organism were not uncommon. Thus high-field NMR (originally 200 MHz and then up through 600-800 MHz), mass spectrometry that involved MS-MS techniques, and chromatographic methods of all types were used. It should be emphasized that HPLC, the use of which is effectively a sina qua non in modern isolation methods, was not generally available until the late 1970s, and thus isolations often required large amounts of materials due to the level of sophistication of the techniques available. In retrospect, this is one of the major reasons that the field evolved slowly. Discovery of a given compound was easy in relative terms, but development, which required large amounts, was, and still is, not simple, as will be shown in examples later in the review. Although Paul Scheuer at the University of Hawaii was the first (marine) natural products chemist to systematically explore the chemistry of marine invertebrates, from his original work in Hawaii in the 1950s until his death † Dedicated to the late Dr. D. John Faulkner (Scripps) and the late Dr. Paul J. Scheuer (Hawaii) for their pioneering work on bioactive marine natural products. * To whom correspondence should be addressed. Tel: (301) 846-5387. Fax: (301) 846-6178. E-mail: dn22a@nih.gov. 1216 J. Nat. Prod. 2004, 67, 1216-1238 10.1021/np040031y This article not subject to U.S. Copyright. Published 2004 by the Am. Chem. Soc. and the Am. Soc. of Pharmacogn. Published on Web 06/11/2004

early in 2003, initially investigating marine toxin structures, the work of Rinehart at the University of Illinois at Champaign-Urbana and of Pettit at Arizona State University led the way in the discovery of biologically active molecules (i.e., potential human use pharmaceutical agents) from the marine environment. Both of these research groups were funded by the U.S. government but in somewhat different ways in the beginning. Pettit was part of an antineoplastic drug discovery effort whereby organisms were collected and extracted by NCI-funded groups, and Table 1. Status of Marine-Derived Natural Products in Clinical and Preclinical Trials name source status (disease) comment didemnin B Trididemnum solidum Phase II (cancer) dropped middle 90s dolastatin 10 Dolabella auricularia (marine microbe derived; cyanophyte) Phase I/II (cancer) many derivatives made synthetically; no positive effects in Phase II trials; no further trials known girolline Pseudaxinyssa cantharella Phase I (cancer) discontinued due to hypertension bengamide derivative Jaspis sp. Phase I (cancer) licensed to Novartis, Met-AP1 inhibitor, withdrawn 2002 cryptophycins (also arenastatin A) Nostoc sp. & Dysidea arenaria Phase I (cancer) from a terrestrial cyanophyte, but also from a sponge as arenastatin A; synthetic derivative licensed to Lilly by Univ. Hawaii, but withdrawn 2002 bryostatin 1 Bugula neritina Phase II (cancer) now in combination therapy trials; licensed to GPC Biotech by Arizona State Univ.; may be produced by bacterial symbiont TZT-1027 synthetic dolastatin Phase II (cancer) also known as auristatin PE and soblidotin cematodin synthetic derivative of dolastatin 15 Phase I/II (cancer) some positive effects on melanoma pts in Phase II; dichotomy on fate ILX 651, synthatodin synthetic derivative of dolastatin 15 Phase I/II (cancer) in Phase II for melanoma, breast, NSCLC ecteinascidin 743 Ecteinascidia turbinata Phase II/III (cancer) in 2003 licensed to Ortho Biotech (J&J); produced by partial synthesis from microbial metabolite aplidine Aplidium albicans Phase II (cancer) dehydrodidemnin B, made by total synthesis E7389 Lissodendoryx sp Phase I (cancer) Eisai’s synthetic halichondrin B derivative discodermolide Discodermia dissoluta Phase I (cancer) licensed to Novartis by Harbor Branch Oceanographic Institution kahalalide F Eylsia rufescens/ Bryopsis sp. Phase II (cancer) licensed to PharmaMar by Univ. Hawaii; revision of structure ES-285 (spisulosine) Spisula polynyma Phase I (cancer) Rho-GTP inhibitor HTI-286 (hemiasterlin derivative) Cymbastella sp Phase II (cancer) synthetic derivative made by Univ. British Columbia; licensed to Wyeth KRN-7000 Agelas mauritianus Phase I (cancer) an agelasphin derivative squalamine Squalus acanthias Phase II (cancer) antiangiogenic activity as well Æ-941 (Neovastat) shark Phase II/III (cancer) defined mixture of <500 kDa from cartilage; antiangiogenic activity as well NVP-LAQ824 Synthetic Phase I (cancer) derived from psammaplin, trichostatin, and trapoxin structures Laulimalide Cacospongia mycofijiensis preclinical (cancer) synthesized by a variety of investigators Curacin A Lyngbya majuscula preclinical (cancer) synthesized, more soluble combi-chem derivatives being evaluated vitilevuamide Didemnum cucliferum & Polysyncraton lithostrotum preclinical (cancer) diazonamide Diazona angulata preclinical (cancer) synthesized and new structure elucidated eleutherobin Eleutherobia sp. preclinical (cancer) synthesized and derivatives made by combichem; can be produced by aquaculture sarcodictyin Sarcodictyon roseum preclinical (cancer) (derivatives) combi-chem synthesis performed around structure peloruside A Mycale hentscheli preclinical (cancer) salicylihalimides A Haliclona sp. preclinical (cancer) first marine Vo-ATPase inhibitor; similar materials from microbes, synthesized thiocoraline Micromonospora marina preclinical (cancer) DNA polymerase R inhibitor ascididemnin preclinical (cancer) reductive DNA-cleaving agents variolins Kirkpatrickia variolosa preclinical (cancer) Cdk inhibitors dictyodendrins Dictyodendrilla verongiformis preclinical (cancer) telomerase inhibitors GTS-21 (aka DMBX) Phase I (Alzheimer’s) modification of a worm toxin; licensed to Taiho by Univ. Florida manoalide Luffariaella variabilis Phase II (antipsoriatic) discontinued due to formulation problems IPL-576,092 (aka HMR-4011A) Petrosia contignata Phase II (antiasthmatic) derivative of contignasterol; licensed to Aventis IPL-512,602 derivative of 576092 Phase II (antiasthmatic) with Aventis IPL-550,260 derivative of 576092 Phase I (antiasthmatic) with Aventis ziconotide (aka Prialt) Conus magus Phase III (neuropathic pain) licensed by Elan to Warner Lambert CGX-1160 Conus geographus Phase I (pain) contulakin G CGX-1007 Conus geographus Phase I (pain & epilepsy) conantokin G; discontinued AMM336 Conus catus preclinical (pain) ω-conotoxin CVID ø-conotoxin Conus sp. preclinical (pain) conotoxin MR1A/B CGX-1063 Thr10-contulakin G preclinical (pain) modified toxin ACV1 Conus victoriae preclinical (pain) R-conotoxin Vc1.1 Reviews Journal of Natural Products, 2004, Vol. 67, No. 8 1217

the extracts tested by NCI contractors for their ability to inhibit the growth of tumors in mice. The active principles were then isolated by NCI-funded groups by following the bioactivity in mice. This was probably the first large-scale application in the marine area of what has come to be known as “bioactivity-driven isolations”. Rinehart, however, was funded by a number of U.S. government agencies but initially used his MS-MS and NMR capabilities to determine the potential structures of the bioactive agents that he found in organisms collected predominately in the Caribbean during NSF-funded expeditions. There have been a number of recent reviews covering aspects of this area, either not in as much detail or from a clinical or preclinical aspect. The reader should consult them for comparative purposes;6-13 any reviews that are specific to a class of agents will be cited under the agents themselves. We will discuss agents by clinical activities rather than by source or chemical class, and in order to aid the reader, we have shown in Table 1 all of the sources, diseases, trial level achieved at date of review submission, and a short comment where necessary on the compounds that we discuss in the review. The order is by type of pharmacological activity and then clinical and/or preclinical results for each activity, with those that have been discontinued listed first in each disease. Introduction to Agents that Entered Antitumor Clinical Trials The significant number of compounds from marine sources that have been entered into antitumor preclinical and clinical trials since the early 1980s is due to two serendipitous findings. The first is that the agents elaborated by marine organisms must be affected by the dilution effects of seawater; thus any “chemical warfare” agent must be extremely potent, as it has to overcome dilution en route to its target. This process may be considered as analogous to the role of phytoalexins in the plant kingdom, or similar to the emission of pheromones by insects, though the purpose in the latter case is to attract rather than repel! The other is that the U.S. National Cancer Institute (NCI) has funded, either directly or indirectly, most of the search for agents active against cancer, irrespective of the source. Thus, one has the systems in place for collection, bioactivity determinations, and subsequent testing in animals and humans, with the aim of finding new and potent treatments for cancers. Because of the extremely long time frame involved in such processes (for example, paclitaxel (Taxol), took over 20 years from structural determination and reporting until FDA approval in the early 1990s), the compounds that will be discussed fall into two approximate time frames: those from the initial collection programs (which aided the didemnin B discovery vide infra) and those that are further back in the current system that have been discovered as a result of the modified NCI screens utilizing the 60 cell line (or functional equivalent) screen that has been in use from the early 1990s. Some of the agents whose mechanisms of action (MOA) were discovered as a result of the latter screening system are now either just entering or about to enter clinical trials. Agents Now Withdrawn from Antitumor Clinical Trials Didemnin B. This compound (4) was isolated by Rinehart’s group from extracts made of the tunicate Trididemnum solidum14 that demonstrated excellent antiviral activity and subsequent cytotoxic activity against P388 and L1210 murine leukemia cell lines. Didemnin B was advanced into preclinical and clinical trials (Phases I and II; see Table 3 in Nuijen et al. for a discussion of these trials13) under the auspices of the NCI in the very early 1980s as the first defined chemical compound directly from a marine source to go into clinical trials for any major human disease. Despite many different treatment protocols and testing against many types of cancer, the compound turned out to be too toxic for use, and trials were officially terminated in the middle 1990s by NCI. Even though this compound did not make it to Phase III trials and then to market, the experience gained from these efforts was immensely helpful in aiding the trials of other natural product-derived agents/compounds. Thus Rinehart’s group developed methods of large-scale isolation and purification and, as would become essential much later in time, total syntheses that permitted significant structureactivity relationships to be derived.15 This work permitted materials to be provided to others so that basic biochemical studies could be performed, leading to the identification of a potential MOA for this compound, with the binding to elongation factor 1-R (ef1-R) being reported by Crews et al. in the middle 1990s.16 Subsequent reports from Crews’ group showed that didemnin B binds noncompetitively to palmitoyl protein thioesterase,17 and the following year, Johnson and Lawen reported that rapamycin inhibited the didemnin-induced apoptosis of human HL-60 cells, perhaps by binding to the FK-506 binding protein(s).18 Inferentially, from this latter result, didemnin B might bind to or modulate the FK-binding proteins as part of its immunomodulatory process and thus lead to cell death via apoptosis. Table 2. Phase I and Phase II Combination Studies with Bryostatin 1 year phase schedule dose range tumor type(s) # pts CR PR SD side effects reference 2001 I 24 h infusion & bolus of vincristine, dose escalation of bryostatin, 1-5 cycles 12.5-62.5 µg‚M2 bryostatin; 1.4 mg‚M2 of vincristine B-cell cancer 25 1 2 4 myalgia; neuropathy Dowalti et al.324 2002 I 24 h infusion, days 1 & 11, AraC on days 2, 3, 9, 10, bryostatin dose escalation, fixed AraC, 1-6 cycles 12.5-50 µg‚M2 bryostatin; 1-3 gm‚M2 AraC leukemia 23 5 1a 0 myalgia; neutropenia Cragg et al.325 2002 I 24 h infusion, fludarabine for days 2-6, repeat at 28d, or reverse addition order, 6-9+ cycles 16-50 µg‚M2 bryostatin; 12.5-25 mg‚M2 FAra CLL; NHL 53 bb b neutropenia Roberts et al.326 2003 II 1 h infusion of paclitaxel on 1, 8, & 15d; 24 h infusion of bryostatin on 2, 9, 16d, repeated on 28d cycle, 1-4 cycles 40-50 µg‚M2 bryostatin; 90 mg‚M2 paclitaxel NSCLC 11 0 2a 5 myalgia Winegarden et al.327 a Some question as to response level. b 23 “nondefined objective responses”. 1218 Journal of Natural Products, 2004, Vol. 67, No. 8 Reviews

In 2002, Vera and Joullie19 published an excellent review of didemnins as cell probes and targets for syntheses and also made some reasonable arguments that the dosing schedules used in the early clinical trials may well have been nonoptimal for demonstrating activity as a cytotoxin rather than as an immunosuppressive/modulator. It will be interesting to compare the dosing schedules and responses for didemnin B and aplidine (Aplidin; PharmaMar, vide infra) in man once the latter are fully reported in the literature. Although didemnin B was not successful, a very close chemical relative is currently in clinical trials (cf. aplidine below), and in 2000 Rinehart published an overview of these compounds as part of a discussion of antitumor compounds from tunicates, which the reader may consult for further details.20 Dolastatin 10. The dolastatins are a series of cytotoxic peptides that were originally isolated in very low yield from the Indian Ocean mollusk Dolabella auricularia by Pettit’s group as part of its work on marine invertebrates.21-25 Due to the potency and mechanism of action of dolastatin 10 (5), a linear depsipeptide that was shown to be a tubulin interactive agent binding close to the vinca domain at a site where other peptidic agents bound,26,27 the compound entered Phase I clinical trials in the 1990s under the auspices of the NCI. Since the natural abundance was so low, Pettit and others developed synthetic methods that provided enough material under current Good Manufacturing Practice (cGMP) conditions to allow clinical trials to commence.25 Dolastatin 10 progressed to Phase II trials as a single agent, but although tolerated at the doses used, which were high enough to give the expected levels in vivo to inhibit cell growth, it did not demonstrate significant antitumor activity in a Phase II trial against prostate cancer in man.28 Similarly, no significant activity was seen in a Phase II trial against metastatic melanoma, even though again, levels high enough to affect cells were demonstrated.29 There are other dolastatins and molecules related to them that are still in clinical and preclinical trials; they will be covered in later sections. Girolline (Girodazole). This very simple compound, a substituted imidazole (6), was reported from the sponge Pseudaxinyssa cantharella30 and was shown by workers at Rhone-Poulenc Rorer to be an inhibitor of protein synthesis, acting preferentially on the termination step in eukaryotic protein synthesis, in contrast to other known protein synthesis inhibitors such as emitine, homoharringtonine, anguidine, and bruceantin, which generally act at either the initiation or elongation steps.30 Girolline proceeded to Phase I clinical trials in man, but the trials were stopped due to significant hypertensive effects seen in treated patients. In 2002, Schiavi et al. 31 reported on the synthesis of one of the two possible thiazole derivatives of girolline, 5-deazathiogirolline (7), hoping that this simple substitution might alter the human toxicity characteristics. Although protected intermediates in the synthetic scheme were about 10% as active in girolline in comparable systems, the final deprotected product (the thiazole derivative) was effectively inert. Bengamide Derivatives. Bengamides A (8) and B (9) were first reported in 1986 as antihelminthic compounds (together with some antibiotic and cytotoxic activites) by Crews’ group at the University of California, Santa Cruz.32 The number of bengamide analogues isolable from the same sponge was extended to bengamide G, with details being reported on their isolation and absolute stereochemistry in two more papers from the same group.33,34 In a subsequent paper with workers from Novartis, the number of compounds in the group was extended, and their antitumor activities were reported.35 The bengamides were evaluated by Novartis (initially by Ciba-Geigy), as Ciba-Geigy was the then current industrial partner of the UCSC group in an NCI-funded National Cooperative Natural Products Drug Discovery Group (NCNPDDG). As a result of their intrinsic activities, a synthetic program was put in place that developed a derivative of bengamide A (10) as a clinical candidate. This derivative was shown to be an inhibitor of methionine aminopeptidases and subsequently entered Phase I clinical trials in 2000, but was withdrawn in the middle of 2002. Cryptophycins. These compounds were reported from two blue-green algae, initially by a group from Merck in 1990 using a Nostoc species (ATCC 53789) originally isolated from a lichen on a Scottish Island; they reported only the antifungal activity, finally deciding not to proceed with development, as it was too toxic. Moore’s group at the University of Hawaii then identified the same compound36 from a nonmarine cyanophyte, Nostoc sp. strain GSV-224, Reviews Journal of Natural Products, 2004, Vol. 67, No. 8 1219

and in addition, almost contemporaneously, a similar molecule was reported by Kobayashi et al. from an Okinawan sponge (see below). The University of Hawaii and Wayne State University licensed the natural cryptophycins and synthetic derivatives to the Lilly Company for advanced preclinical and clinical development. This led to the selection of cryptophycin 52 (LY355703) (11) as a Phase I clinical candidate in the middle 1990s, with a single publication37 in late 2002 giving the Phase I and pharmacological results from a variety of schedules, with an intermittent schedule being chosen for Phase II studies. The routes, both chemical and pharmacological, leading to the choice of this particular derivative were described by Shih and Teicher38 of the Lilly Research Laboratories. The compound progressed toward Phase II trials, but in 2002, cryptophycin 52 was withdrawn from trial (personal communication, Dr. R. Moore). Although the original cryptophycins came from terrestrial cyanophytes and the clinical candidate came from semisynthetic modifications of the natural product, in 1994 Kobayashi et al. reported that an acetone extract of the Okinawan sponge Dysidea arenaria had potent cytoxicity,39 and on purification, the compound arenastatin A (12) subsequently turned out to be identical to cryptophycin 24 (12) reported by Moore’s group in 1995.40,41 A later report from the Japanese group42,43 demonstrated that arenastatin A and synthetic analogues also are tubulin interactive agents similar in activity to the other cryptophycins reported by Moore et al. Agents Currently in Clinical Trials as Antitumor Agents Bryostatins. In 1968, NCI commissioned a large-scale (for those days) collection of the bryozoan Bugula neritina by Jack Rudloe of the Gulf Specimen Company off the west coast of Florida that was sent to Pettit’s group for chemical workup. The aqueous 2-propanol extract was subsequently tested by NCI for its intrinsic activity as an antitumor agent in the then current P388 and L1210 murine leukemia in vivo models. Subsequently, the extract was found to be inactive against L1210 but to give a 68% increase in life span using P388 at the same concentration.44 Following significant amounts of work by Pettit and his group, including more collections on a larger scale, significant problems with isolation as a result of dealing with vanishingly small quantities of a very potent agent, and problems related to assay reproducibility, the compound was purified and identified as bryostatin 3 (13), one of a series of closely related compounds that now number 20.44-49 Subsequent work by Pettit’s group identified two other geographic areas where significant (in relative terms) quantities of bryostatin 1 (14) could be isolated from B. neritina colonies. What is important, however, is that although a number of reports have been made about other taxa producing bryostatins, in almost all cases, on careful examination, the putative producing organism actually contains B. neritina. However, as a result of prodigious efforts on the part of Pettit and collaborators and workers at NCI-Frederick, by 1990 there was enough cGMP-grade material to commence systematic clinical trials, though prior to this time frame, small quantities of bryostatin 1 had been supplied to a variety of collaborators so that basic biochemical studies and initial clinical trials in the U.K. could be performed. From these studies, which are summarized in recent reviews by a number of authors,48-50 it was shown that bryostatins bind to the same receptors as the tumorpromoting phorbol esters, the protein kinase C (PKC) isozymes, but have little or no tumor promoter activity. A recent paper from Hale’s group51 where they made a modified analogue has shown that the binding site for this compound and, by inference, the bryostatins is almost certainly at the cysteine-rich domain 2 (CRD2) in human PKC-R. As a result of this binding, the PKC isozymes in various tumor cells are significantly down-regulated, leading to inhibition of growth, alteration of differentiation, and/or death. To date, bryostatin 1 has been in more than 80 human clinical trials, with more than 20 being completed at both the Phase I and Phase II levels. There have been some responses to the compound as a single agent with effects ranging from complete remission (CR), to partial remission (PR), to stable disease (SD). However, the use as a single agent is probably not the optimal usage for this compound. More detailed reports of the clinical development are given in the recent reviews by Pettit50 and by Clamp and Jayson.52 However, when bryostatin is combined with another cytotoxin, such as the vinca alkaloids or nucleosides, and the carcinomas are leukemic in nature, then the response rates, even in Phase I trials, begin to demonstrate that such mixed treatments may well be worth further investigation (cf. Table 2 for details/citations). Thus a combination with high levels of AraC and low levels of bryostatin in patients with leukemias, in a population that included patients who had failed high-dose AraC (HiDaC) therapy, five of 23 patients presented with complete responses in a recent Phase I trial. Similarly, patients with chronic lymphocytic leukemia (CLL) and nonHodgkins lymphoma (NHL) treated with fludarabine and bryostatin were reported to show close to 50% “objective responses” in the trial report. With non-small cell lung cancer (NSCLC) and paclitaxel/bryostatin, seven of 11 1220 Journal of Natural Products, 2004, Vol. 67, No. 8 Reviews