Reviews Journal of Natural Products.2004.Vol.67.No.812 se (ke tures but re ed a sic mificant amount of the web site http:/ heeolecaeisoipated nuloc hage-colony facto ted phorbol es with the figures being in cladribin (15.)had acti n in vitro cell to Dactaxelingistineiorcdplatin from these ere mad due n 8 nM bi (15)to nity ED to the oa.o struc shown) a most all cases repor this was treatable by stand nM range depending upon the fatty acid used. the resu the early zation that For ex to obt with 13 met e en p statin I ab ove SIM)in order t h the par ters i d through the de ne.The en ed h ndola se and this re ulted fo on as to method mo aget ryostatins re the 20 The Evans catio n where by thy nd 15 n200 .the gh 2002.o the ym and me 11m ell line s.Finally. e en 0f2 ulted for 03.he p tio n of theth red to abov further r ments of the model c dd)with ne ne v inte ing question sing from the s arch nt However. ah one size nism 05 tion of such an agent might well be a viable option. ies that act lly produ able tin tial bnding s questio n the work of H gues of thes graphy.Hayg an is ac bryostatin e th s that utative would maintain the putative binding sites at the oxygen polyketide synthase (PKS)gene fragment in the microbia
patients in a Phase II trial demonstrated positive responses (PR/SD) but no CRs. Currently (01/2004), there are four Phase I and five Phase II trials underway (data from the NCI clinical trials web site http://clinicaltrials.gov), and in every case, these are combination studies with biologicals such as interleukin 2 or granulocyte macrophage-colony stimulating factor (GM-CSF), nucleoside derivatives such as gemcitabine, cladribine, or AraC, or other cytotoxic agents such as paclitaxel, vincristine, or cisplatin. These combinations are being tested against leukemias and lymphomas and ovarian and prostate carcinomas. Hopefully, results similar to those demonstrated in Table 2 will be reported in due course. In all of the clinical trials so far reported the major cause of dose-limiting toxicity (DLT) appears be myalgia, but in almost all cases reported this was treatable by standard supportive therapies and patients continued on trial. Details as to the protocols for all trials and the results reported are given in two articles currently in press.53,54 A very interesting “side effect” of the use of bryostatin as a clinical candidate was the early realization that wild collections would not suffice to produce enough of the material for use as a clinical entity. For example, to obtain enough material for the initial clinical trials under NCI auspices, it was necessary to begin with 13 metric tonnes55 of wild-collected B. neritina and then process the material using large-scale chromatographic techniques in order to produce 18 g of cGMP bryostatin 1. Subsequently, NCI funded in-sea and on-land aquaculture (total NCI expenses above $1M) in order to establish the parameters necessary to produce bryostatin 1 in sufficient quantities at a “reasonable” cost if it progressed through the development pipeline. The processes involved and the successful results have recently been reviewed by Mendola,56 and this review should be consulted for specific information as to methods, economics, etc. Since the publication of the first structure by Pettit in 1982, these molecules have been the target of many synthetic chemistry groups. Many partial syntheses have been published where specific portions of the molecule have been made, but to date, only three of the 20 reported bryostatins have been synthesized. The first was the enantioselective total synthesis of bryostatin 7 in 1990 by Masamune et al.,57 the second by Evans et al. on the enantiomeric total synthesis of bryostatin 2 in 1999,58 and the third, the synthesis of bryostatin 3, by the group of Nishiyama and Yamamura59 in 2000. In addition to these papers, three excellent review articles have been published covering information available through 2002, on the syntheses of these three and other partial bryostatin structures including bryostatin 1, and should be consulted for specific details of reaction schema and comparisons of routes.48,49,60 From inspection of the three reviews referred to above it can be stated that the total synthesis of bryostatin 1 is not the process that one would wish to utilize to produce this agent. However, if one could synthesize a simpler analogue with comparable activity, then chemical production of such an agent might well be a viable option. In 1986, Wender et al. analyzed the potential binding site of the phorbol esters on PKC as a guide to the design of simpler analogues of these agents.61 In 1988, this work was expanded by modeling bryostatin 1 onto the same binding site as a result of the initial results indicating that bryostatin 1 interacted with PKC.62 Subsequently, the modeling work was refined to produce three analogues that would maintain the putative binding sites at the oxygen atoms at C1 (ketone), C19 (hydroxyl), and C26 (hydroxyl) in the original molecule. These requirements gave rise to structures (15-17) that maintained the recognition features but removed a significant amount of the peripheral substituents. These molecules demonstrated nanomolar binding constants when measured in displacement assays of tritiated phorbol esters, with the figures being in the same general range as bryostatin 1, and two compounds (15, 16) had activities in in vitro cell line assays close to those demonstrated by bryostatin 1 itself.63-66 Following on from these examples, modifications were made to the base structure (15) to introduce a second lactone (18) that had an 8 nM binding affinity and also inhibited P388 with an ED50 of 113 nM.67 Concomitantly, modifications were made to the base analogue (15) where different fatty acid esters were made (structures not shown). These, too, exhibited binding affinities for PKC isozymes in the 7-232 nM range depending upon the fatty acid used.68 To show the versatility of the base structure, recently Wender published a simple modification where by removal of a methyl group in the C26 side chain in compound 15 to produce compound 19, the binding affinity to PKC was increased to the picomolar level,69 and the compound demonstrated greater potency than bryostatin 1 in in vitro cell line assays. Finally, at the end of 2003, he published improved syntheses of the molecules, which could permit further rapid improvements of the model compound(s) with the potential for much greater overall yields.70,71 One very interesting question arising from the search for bryostatin sources was, why is the nominal producing organism so ubiquitous, but the number of B. neritina colonies that actually produce detectable bryostatin 1-3 levels so low and geographically spread? One possible answer to this question came from the work of Haygood and her collaborators at the Scripps Institution of Oceanography. Haygood showed that the bryozoan is actually the host to a symbiotic microorganism that may well be the actual producer of the compound; in an elegant series of experiments, she and her colleagues demonstrated by use of molecular probes the presence of a putative type I polyketide synthase (PKS) gene fragment in the microbial Reviews Journal of Natural Products, 2004, Vol. 67, No. 8 1221
1222 Journal of Natural Products,2004.Vol 67.No.8 Reviews aygood demonstrated that th nt depths.Thus,at dept nents are found m ese are also kn oducer than 9 m(S or only the min HHH ic micr 。。。 ity ar 21 Dolastatin todin (LU-103793).An biol t by of and other The ate the n tri al Marine Bioe chnolog gtoiciesbeertedlatthePha numb of the the current sta trials used a very rapid bolus (5m in iv rch. tha infusin:from them the invest erubeetle's p oal symbiont PKS that und pro sed into Phas hat an A doma tive respon mains ollo the tri Havgood is su of the organism the as a su the supply if bryostatin become literature as s comp us Amad et al Jun (01/2004)listed as discontinued in the Prous Ensemble database. 。。 20 0天 。。。 22 arlier. of the dos ns hav Dolastatin Derivative nadotin).Ther a in of eithe agent (22) ll as pres see.Daiichi Phar ancer Re arch (AACR)meeting. or the joint US n in abstract form orally acti Recently.a further e mock trans mors in Phase I patients in 15 ha early or adva xenografts at levels o s or to b useful "biopro bulin int actior tive and com etastatin was not,even t500 cently repo d tha t the vinc ain in tubulin thg-kg may wehan bei of a a si of competition were found Very recentthe a fthe labe compound to prostate cancer cells.by using the up with the bryostatins.there was always a potential question
flora of colonies that produced bryostatin but that was absent in the corresponding flora of nonproducers.72 In addition, Davidson and Haygood demonstrated that there are subdivisions within B. neritina samples taken from the same sites but at different depths. Thus, at depths greater than 9 m (the D or deep type), bryostatins 1-3 and minor components are found (these are also known as producers of chemotype O for “octa-2,4-dienoatic chain”), whereas, at less than 9 m (S or shallow type), only the minor derivatives are seen (chemotype M). The symbiotic microbes (Candidatus Endobugula sertula) isolated from each type differ in their mitochondrial carboxylase I (CO I) sequences by 8%, giving rise to the possibility that the bryozoans are also different taxonomically.73 There were reports at the Society for Industrial Microbiology (SIM) meetings in 2002 and 2003 that demonstrated that Haygood and collaborators were pursuing the possibility of transferring this particular PKS fragment to other, more amenable microbes in order to further investigate the possibility of producing bryostatin by fermentation. At the recent 6th International Marine Biotechnology Conference (IMBC) in Chiba, Japan, Haygood74 reported on the current status of the PKS search, suggesting that this system resembled that reported by Piel75,76 for the Paederus beetle’s pseudomonal symbiont PKS that produces pederine, in that there are no acyl-transferase (AT) domains in the clusters, unlike the usual PKS system, but that an AT domain was found in another, more remote, area of the overall PKS system. Further work is ongoing utilizing such “remote” AT domains from another organism. It will be very interesting to follow these results if Haygood is successful, as cultivation of the organism, or a surrogate with the bryostatin PKS system expressed, would potentially solve any supply problems if bryostatin becomes a commercial drug. Dolastatin Derivative, TZT-1027 (Auristatin PE or Soblidotin). As a result of the synthetic processes alluded to earlier, many derivatives of the dolastatins have been synthesized with TZT-1027 (20), now in Phase I clinical trials in Europe, Japan, and the United States under the auspices of either Teikoku Hormone, the originator, or the licensee, Daiichi Pharmaceuticals. This compound is also known as Auristatin PE and Soblidotin, and an initial report on Phase I studies was given in abstract form77 at the American Society for Clinical Oncology (ASCO) in 2002. Recently, a further report from investigators at Teikoku Hormone indicated that in nude mice the transfected vascular endothelial growth factor (VEGF)-secreting human lung cell line SBC-3/VEGF and also the mock transfected cell line were effectively totally inhibited as either early or advanced stage xenografts at levels of 1 or 2 mg‚kg-1, conditions under which only vincristine was similarly active and combretastatin was not, even at 500 mg‚kg-1. What was of significant interest in addition to these results was that TZT-1027 also exhibited a potent antivascular effect at these levels, thus suggesting that a dual mechanism might well be possible with this agent.78 Very recently, a multinational group of investigators demonstrated the potential for a directed delivery of this compound to prostate cancer cells, by using the upregulation of the adhesion molecule, E-selectin, that is found in the epithelium of prostate carcinomas and demonstrated that a monoclonal antibody directed to this protein with auristatin linked via a cathepsin B-labile linker gave more than 85% inhibition of growth of prostate carcinoma cell lines in mouse models.79 Dolastatin Derivative, Cematodin (LU-103793). Another derivative of dolastatin 15 known as Cematodin (21) (and also as LU-103793) was placed into Phase I clinical trials by BASF Pharma under their Knoll division for treatment of breast and other cancers. The results from six trials have been reported at the Phase I level with doselimiting toxicities being neutropenia or cardiotoxicity. A number of these trials used a very rapid bolus (5 min iv), and others used a longer time frame, even up to 5 days of continuous infusion; from them, the investigators’ recommended ranges for Phase II studies were at the 2.5-10 mg‚ M2 dose levels.80-85 The compound progressed into Phase II studies against malignant melanoma, metastatic breast cancer, and non-small-cell lung cancer, and reports demonstrated no objective responses in any of the trials86-88 although stable disease was seen in both the melanoma and breast cancer trials and there was a subjective increase in a quality of life measure in the lung trial. Currently, there is some dichotomy in the literature as to whether work is actively continuing with this compound; thus Amador et al. report Phase II trials still ongoing as of June 2003 in breast, ovarian, lung, prostate, and colon carcinomas,12 whereas it is now currently (01/2004) listed as discontinued in the Prous Ensemble database. Dolastatin Derivative, ILX651 (Synthadotin). There have been six scientific reports in the last two years on the Phase I studies with this agent (22), all as presentations at ASCO meetings,89-92 the American Association for Cancer Research (AACR) meeting,93 or the joint USEuropean (AACR-NCI-EORTC) molecular targets meeting.94 ILX651 is an orally active third generation dolastatin 15 derivative that was licensed by Ilex from BASF Pharma, and two reports95,96 in 2003 indicated that Ilex Oncology is initiating Phase II studies in melanoma, breast, and nonsmall-cell lung cancers, as there were responses in these tumors in Phase I patients. From a nonclinical perspective, dolastatin 15 has proven to be a useful “bioprobe” in tubulin interaction studies. Thus, by using tritium-labeled dolastatin 15, Hamel’s group at NCI97 recently reported that the vinca domain in tubulin may well be composed of a series of overlapping domains rather than being a single entity, as different levels/types of competition were found when selected tubulin interactive agents were used to investigate the binding characteristics of the labeled probe. Source(s) of the Dolastatins. Similarly to the situation with the bryostatins, there was always a potential question 1222 Journal of Natural Products, 2004, Vol. 67, No. 8 Reviews
Reviews Journal of Natural Products.2004.Vol.67.No.8 1223 with the dolastatins as to whether they were microbial in with Et743.110 Subsequently,he improved the synthetic origin,as peptides with unusual amino acids have been schema and developed a refined process that produced both well documented in the literature as coming from the Et743 and phthalascidin at much higher yields.111 Other Cyanophyta.In the past few years,this supposition has synthetic chemistry groups have continued work on the been shown to be fact.Thus,in 1998,workers at the basic compound,but as yet,none of their compounds have Universities of Guam and Hawaii reported98 the isolation had any biological activity reported in the literature.2. and purification of simplostatin 1(23)from the marine The natural compound was licensed by the University cyanobacterium Simploca hynoides.This molecule differed of Illinois to the Spanish Company PharmaMar for subse- from dolastatin 10 by the addition of a methyl group on quent development.Following very large-scale wild collec- the first N-dimethylated amino acid.Subsequently,in 2001 tions and aquaculture on both land and in-sea in efforts to the same groups reported the direct isolation of dolastatin obtain enough source material for further preclinical and 10 from another marine cyanobacterium that was known clinical workup,PharmaMar chemists performed an el- to be grazed on by D.auricularia.99 Dolastatin 10 was in egant semisynthesis from the marine Pseudomonas fluo fact isolated from the nudibranch following feeding of the rescens metabolite cyanosafracin B that provided cGMP cyanophyte,thus confirming the original hypothesis (per- grade Et743 from a 21-step synthetic process on a scale sonal communication.Dr.V.J.Paul). large enough to provide enough material for clinical trials. Very recently,the MOA of symplostatin 1 was evaluated This was feasible despite a low overall vield of 1.4%because both in vitro and in vivo,and it was shown to be similar to the starting material could be obtained on a large scale by dolastatin 10 but to be somewhat more toxic to mice at fermentation.The original paper114 together with a rela- comparable doses.100 In addition,two further examples of tively recent review article,115 both from the PharmaMar dolastatin-like peptides isolated from different collections group,should be consulted for further details as to syn- of the ubiquitous cyanophyte Lyngbya majuscula have thetic strategies,etc.,employed for production of this recently been reported in the literature,viz.,dolastatin 16 compound. from a Madagascan collection by Nogle and Gerwick101 and A number of reports have been published in the litera- homodolastatin 16 from a Kenyan collection by Davies- Coleman et al.,102 further evidence for the microbial source ture over the past few years giving possibilities as to the MOA(s)of Et743 when tumor cells are treated in vitro.A of these peptidic cytotoxins. significant problem with some of the reports is that the concentration(s)used in the experiments are orders of magnitude greater than those that demonstrate activity in vitro.These levels are in the low nanomolar to high picomolar range,and thus care should be taken when evaluating published work on the MOA of this compound. At physiologically relevant concentrations the MOAs of Et743 have been shown to include the following:effects on the Transcription-coupled Nucleotide Excision Repair process(TC-NER)116.117 and interaction between the Et743 DNA adduct and DNA transcription factors,in particular the NF-Y factor.118 In the recent review on Et743 by a Dutch group,19 further details as to other possible mech- anisms are given in their Table 1;the references that they Ecteinascidin 743.Antitumor activity from the ascid cite should be consulted for in-depth information and ian Ecteinascidia turbinata had been reported as early as discussion for other potential MOAs ascribed to Et743.As 1969 by Sigel et al.,103 but it was not until 1990 that the addenda to the results given in the paper above,there were structures of the active components were published simul- two presentations at the AACR-NCI-EORTC molecular taneously by Rinehart et al.104 and Wright et al.105 targets meeting in November 2003 reporting gene expres- The structure of the most stable member of the series. sion profiles on sarcoma lines using the"Oncochip",a 6700 known as Et743 from the absorption maximum,is shown gene array of genes prevalent in cancer cell proliferation. (24).The base structure,without the exocyclic isoquinoline The first,using cells from treated sarcoma patients,120 group,is a well-known chemotype106 originally reported reported that when the ICso values for Et743 were <100 from microbes,where the compound classes are saframy- nM,early changes(within 6 h)in genes related to apop- cins,naphthyridinomycins,safracins,and quinocarcins tosis,cell cycle,transcription factors,growth factors/ Similar molecules were reported from marine mollusks,i.e., receptors,and binding to nucleic acids were demonstrable. jorumycin from the nudibranch Jorunna funebris107 and In contrast,with cells showing ICso values 2100 nM from sponges,the renieramycins,with the latest variation. (nominally resistant to Et743),there was a marked delay renieramycin J,being recently reported by Oku et al.108 in critical regulatory gene changes.In the other presenta- However,with Et743,the exocyclic substituent was novel, tion121 Martinez et al.reported that using human chond as was the bridging sulfur. rosarcoma lines,there was a 5.5%difference between the The yield from natural sources was very low,and in sensitive and an isogenic resistant line,particularly in the order to provide enough material to perform basic studies cyclin D1/D3,GRO1,and NF-kB pathways.As alluded to as to the MOA and in vitro and in vivo animal studies, earlier,although there are a number of other mechanisms significant amounts of the ascidian had to be collected from postulated,on careful inspection,these are usually shown areas around the Caribbean.The compound was synthe- to occur at concentrations of drug well above (i.e.>~250 sized by Coreyl09 in a chemical"tour de force",and as a nM)those that are physiologically relevant.106 result of his synthetic approach,his group also made a The compound was placed into human clinical trials version where the exocyclic ring was a phthalimido sub- while these mechanisms were being worked out,and by situtent.This compound,phthalascidin,demonstrated 2002 it had been in over a 1000 patients in Phase I and significant activity in the same test systems used initially Phase II trialss covering a variety of cancers.Results from
with the dolastatins as to whether they were microbial in origin, as peptides with unusual amino acids have been well documented in the literature as coming from the Cyanophyta. In the past few years, this supposition has been shown to be fact. Thus, in 1998, workers at the Universities of Guam and Hawaii reported98 the isolation and purification of simplostatin 1 (23) from the marine cyanobacterium Simploca hynoides. This molecule differed from dolastatin 10 by the addition of a methyl group on the first N-dimethylated amino acid. Subsequently, in 2001, the same groups reported the direct isolation of dolastatin 10 from another marine cyanobacterium that was known to be grazed on by D. auricularia. 99 Dolastatin 10 was in fact isolated from the nudibranch following feeding of the cyanophyte, thus confirming the original hypothesis (personal communication, Dr. V. J. Paul). Very recently, the MOA of symplostatin 1 was evaluated both in vitro and in vivo, and it was shown to be similar to dolastatin 10 but to be somewhat more toxic to mice at comparable doses.100 In addition, two further examples of dolastatin-like peptides isolated from different collections of the ubiquitous cyanophyte Lyngbya majuscula have recently been reported in the literature, viz., dolastatin 16 from a Madagascan collection by Nogle and Gerwick101 and homodolastatin 16 from a Kenyan collection by DaviesColeman et al.,102 further evidence for the microbial source of these peptidic cytotoxins. Ecteinascidin 743. Antitumor activity from the ascidian Ecteinascidia turbinata had been reported as early as 1969 by Sigel et al.,103 but it was not until 1990 that the structures of the active components were published simultaneously by Rinehart et al.104 and Wright et al.105 The structure of the most stable member of the series, known as Et743 from the absorption maximum, is shown (24). The base structure, without the exocyclic isoquinoline group, is a well-known chemotype106 originally reported from microbes, where the compound classes are saframycins, naphthyridinomycins, safracins, and quinocarcins. Similar molecules were reported from marine mollusks, i.e., jorumycin from the nudibranch Jorunna funebris107 and from sponges, the renieramycins, with the latest variation, renieramycin J, being recently reported by Oku et al.108 However, with Et743, the exocyclic substituent was novel, as was the bridging sulfur. The yield from natural sources was very low, and in order to provide enough material to perform basic studies as to the MOA and in vitro and in vivo animal studies, significant amounts of the ascidian had to be collected from areas around the Caribbean. The compound was synthesized by Corey109 in a chemical “tour de force”, and as a result of his synthetic approach, his group also made a version where the exocyclic ring was a phthalimido subsitutent. This compound, phthalascidin, demonstrated significant activity in the same test systems used initially with Et743.110 Subsequently, he improved the synthetic schema and developed a refined process that produced both Et743 and phthalascidin at much higher yields.111 Other synthetic chemistry groups have continued work on the basic compound, but as yet, none of their compounds have had any biological activity reported in the literature.112,113 The natural compound was licensed by the University of Illinois to the Spanish Company PharmaMar for subsequent development. Following very large-scale wild collections and aquaculture on both land and in-sea in efforts to obtain enough source material for further preclinical and clinical workup, PharmaMar chemists performed an elegant semisynthesis from the marine Pseudomonas fluorescens metabolite cyanosafracin B that provided cGMP grade Et743 from a 21-step synthetic process on a scale large enough to provide enough material for clinical trials. This was feasible despite a low overall yield of 1.4% because the starting material could be obtained on a large scale by fermentation. The original paper114 together with a relatively recent review article,115 both from the PharmaMar group, should be consulted for further details as to synthetic strategies, etc., employed for production of this compound. A number of reports have been published in the literature over the past few years giving possibilities as to the MOA(s) of Et743 when tumor cells are treated in vitro. A significant problem with some of the reports is that the concentration(s) used in the experiments are orders of magnitude greater than those that demonstrate activity in vitro. These levels are in the low nanomolar to high picomolar range, and thus care should be taken when evaluating published work on the MOA of this compound. At physiologically relevant concentrations the MOAs of Et743 have been shown to include the following: effects on the Transcription-coupled Nucleotide Excision Repair process (TC-NER)116,117 and interaction between the Et743 DNA adduct and DNA transcription factors, in particular the NF-Y factor.118 In the recent review on Et743 by a Dutch group,119 further details as to other possible mechanisms are given in their Table 1; the references that they cite should be consulted for in-depth information and discussion for other potential MOAs ascribed to Et743. As addenda to the results given in the paper above, there were two presentations at the AACR-NCI-EORTC molecular targets meeting in November 2003 reporting gene expression profiles on sarcoma lines using the “Oncochip”, a 6700 gene array of genes prevalent in cancer cell proliferation. The first, using cells from treated sarcoma patients,120 reported that when the IC50 values for Et743 were <100 nM, early changes (within 6 h) in genes related to apoptosis, cell cycle, transcription factors, growth factors/ receptors, and binding to nucleic acids were demonstrable. In contrast, with cells showing IC50 values g100 nM (nominally resistant to Et743), there was a marked delay in critical regulatory gene changes. In the other presentation121 Martinez et al. reported that using human chondrosarcoma lines, there was a 5.5% difference between the sensitive and an isogenic resistant line, particularly in the cyclin D1/D3, GRO1, and NF-κB pathways. As alluded to earlier, although there are a number of other mechanisms postulated, on careful inspection, these are usually shown to occur at concentrations of drug well above (i.e. > ∼250 nM) those that are physiologically relevant.106 The compound was placed into human clinical trials while these mechanisms were being worked out, and by 2002 it had been in over a 1000 patients in Phase I and Phase II trials8 covering a variety of cancers. Results from Reviews Journal of Natural Products, 2004, Vol. 67, No. 8 1223