The Chemistry and pharmacology of Taxol® and its derivatives

PHARMACOCHEMISTRY LIBRARY

ADVISORY BOARD

T. Fujita E. Mutschler N.J. de Souza D.T. Witiak F.J. Zeelen

Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan Department of Pharmacology, University of Frankfurt, F.R.G. Research Centre, Hoechst lndia Ltd., Bombay, lndia College of Pharmacy, The Ohio State University, Columbus, OH, U.S.A Organon Research Centre, Oss, The Netherlands

VII

LIST OF CONTRIBUTORS Dr. G. Appendino Dipartimento di Scienza e Tecnologia del Farmaco via P. Giuria 9 10125 Torino ITALY Dr. S.H. Chen Bristol Myers Squibb Pharmaceutical Research Institute P.O. Box 5100 Wallingford, CT 06492-7660 U.S.A.

Dr. L. Landino Chemistry Department University of Virginia Charlottesville, VA 22901 U.S.A. Dr. T. MacDonald Chemistry Department University of Virginia Charlottesville, VA 22901 U.S.A.

Dr. T. Cresteil INSERM U75 Universite Rene Descartes 75730 Paris Cedex 15 FRANCE

Dr. B. Monsarrat Laboratoire de Pharmacologie et Toxicologie Fondamentales CNRS 205 Route de Narbonne 31400 Toulouse FRANCE

Dr. R.C. Donehower Division of Pharmacology and Experimental Therapeutics Johns Hopkins Qncology Center Baltimore, MD 21287 U.S.A.

Dr. E.K. Rowinsky Div. of Pharmacology and Experimental Therapeutics Johns Hopkins Oncology Center Baltimore, MD 21287 U.S.A.

Dr. V. Farina Department of Medicinal Chemistry Boehringer Ingelheim Pharmaceuticals 900 Ridgebury Road Ridgefield, CT 06877 U.S.A.

Dr. I. Royer Laboratoire de Pharmacologie et Toxicologie Fondamentales CNRS 205 Route de Narbonne 31400 Toulouse FRANCE

Dr. D. Gu6nard Institut de Chimie des Substances Naturelles CNRS 91190 Gif-sur-Yvette FRANCE Dr. J. Kant Bristol Myers Squibb Pharmaceutical Research Institute P.O. Box 5100 Wallingford, CT 06492-7660 U.S.A.

Dr. D.M. Was Bristol Myers Squibb Pharmaceutical Research Institute 5, Research Parkway Wallingford, CT 06492-7660 U.S.A. Dr. M. Wright Laboratoire de Pharmacologie et Toxicologie Fondamentales CNRS 205 Route de Narbonne 31400 Toulouse FRANCE

The Chemistry and Pharmacology of Taxol and its Derivatives V. Farina, editor 9 1995 Elsevier Science B.V. All rights reserved

PREFACE Taxol |

a naturally occurring diterpenoid marketed by the Bristol-

Myers Squibb Company, is one of the most exciting antitumor drugs available today. Its current indications (refractory ovarian and metastatic beast cancer) may soon be expanded since the drug is showing activity against lung and head-and-neck cancers. Taxotere |

a closely related analog, is being developed

by Rhone-Poulenc Rorer, and may receive approval this year. Although there are many reasons to be excited about Taxol | there are also reasons to think that chemists can improve upon it. Its low water solubility and the difficulties in formulating it, its lack of activity against certain types of cancer, its numerous clinical side-effects, this drug, all combine to make This book is therefore stimulate further research in

and the rapid emergence of resistance against it a less than optimal chemotherapeutic agent. written for medicinal chemists, in order to this area and to provide the reader with the

necessary background information to start a research program in the area. There are already many reviews on specialized aspects of Taxol | research, as well as two books that cover essentially every possible aspect of research in the field. Therefore, I feel compelled to justify the publication of yet another comprehensive review of the subject, and explain how this work differs from all the other ones. As mentioned above, the book is written mainly for the medicinal chemist, although it should provide useful background to any researcher in the field. I have chosen specific topics that will be relevant in medicinal chemistry research. I will begin by listing the topics that I have not included and why. No historical accounts on the discovery of Taxol | is given here, not because it is not of interest, but because it has been extensively described elsewhere. 1 The "supply problem" is not directly addressed here. It has been widely publicized, especially in the lay press, that supplying all ovarian cancer 1 Wall, M.E.; Wani, M.C. In Taxane Anticancer Agents: Basic Science and Current Status; Georg, G.I.; Chen, T.T.; Ojima, I.; Vyas, D.; ACS Symposium Series; Washington, 1994, p. 18.

patients with Taxol |

would have catastrophic environmental consequences.

At present, the "patient vs. tree" dilemma is no longer an issue, since the core component (a baccatin derivative) from which Taxol | is made can be found in the leaves of the yew tree in high abundance. Other promising fields of research addressing the supply problem, such as plant tissue culture or even fermentation, have been discussed at length elsewhere. 2 For the practicing chemist, the good news is that the baccatins are available commercially, mostly owing to the extensive efforts by Bristol-Myers Squibb and RhonePoulenc Rorer to secure a steady and reliable supply of Taxol | and Taxotere | respectively. With Taxol | soon becoming generic, there will be no lack of companies that will market one or more of the biologically relevant taxanes. Therefore, although the search for alternative methods of taxane production is fascinating and important, these compounds are now becoming available in quantity to researchers. Clinical results are, of course, the endpoint of all pharmaceutical research, and implications obtained from clinical work are discussed throughout the book, especially in chapters 3 and 7. Nevertheless, a detailed account of Taxol | clinical research is not included here, partially because many good accounts have been presented lately, 3 and partially because much of the information contained in these accounts does not help the medicinal chemist in his design of better taxanes. Finally, although scientifically exciting, no account of total synthetic approaches to the taxanes is reported here, for the same reasons given above. Many accounts have been published of the many elegant approaches to Taxol |

and there is no point in duplicating all that large body of information

here. Also, total synthetic efforts have played essentially no role in clarifying the issues of interest to the medicinal chemist, i.e. S t r u c t u r e - A c t i v i t y Relationships (SAR), and will probably contribute very little in the near future, due the structural complexity of these molecules. The present book opens with a review of the naturally occurring taxoids (Chapter 1), written by Prof. Giovanni Appendino. The chapter is not a comprehensive list of all taxoids isolated to date, but attempts a systematic 2 Taxol: Science and Applications; Suffness, M., Ed.; CRC: Boca Raton (in press). 3 Holmes, F.A.; Kudelka, A.P.; Kavanagh, J.J.; Huber, M.H.; Ajani, J.A.; Valero, V. In Taxane Anticancer Agents: Basic Science and Current Status; Georg, G.I.; Chen, T.T.; Ojima, I.; Vyas, D.; ACS Symposium Series; Washington, 1994, p.31.

approach at describing the different classes of taxoids, with particular reference to all skeletal types and the various functionality patterns. Biosynthetic studies are also discussed, as well as some of the basic chemistry and common functionalities of the taxoidic skeleton. Although Taxol | and the baccatins have been the starting materials for the preparation of analogs for SAR studies, this does not have to be true in general and, with a knowledge of the various taxanes available in nature, one can plan the synthesis of compounds that are not readily accessible from the baccatins. In turn, the search for novel compounds of this family may ultimately lead to new antitumor substances. Chapter 2, also by Prof. Appendino, deals with the structural identification of taxoids, mostly by spectroscopic means. The section on NMR spectroscopy contains the first detailed analysis of the influence of structural factors on proton and carbon chemical shifts in taxoids, and therefore should be of extreme utility to workers in the field. The pictorial analysis of 19 1H and 13C spectra of a number of representative taxoids should provide instant help to chemists who are attempting to identify new taxanes (natural or synthetic). Chapter 3, by Dr. Dolatrai Vyas, discusses the formulation of taxanes. After a detailed discussion of the various Taxol |

formulations of possible

clinical relevance, the chapter explores the concept of prodrugs for the purpose of achieving water solubility and bioequivalence with Taxol| The chapter will be useful to all medicinal chemists working on these drugs, as well as on second-generation analogs. Some of the concepts discussed are general enough to be of interest to all medicinal chemists. Chapter 4, By Prof. Michel Wright et al., deals with the metabolism and pharmacokinetics of Taxol| and Taxotere | Knowledge of the fate of a drug in vivo, and specifically its biodistribution, plasma concentration and half-life, as well as inactivation by metabolic transformations and excretion, is extremely important in planning the synthesis of new analogs. New compounds could be specifically prepared to avoid known metabolic processes that lead to inactivation, like side-chain cleavage and oxygenation (hydroxylation) reactions. In Chapter 5, Dr. Shu-Hui Chen and myself give a comprehensive account on the chemistry of taxanes in relation to SAR studies. The SAR field has undergone an explosive growth. In the early 80's, the scarcity of material

as well as of interest in the drug conspired to keep our knowledge of Taxol | SAR to a minimum. The last 4 years have witnessed a frenzy of publications, as academic and industrial laboratories compete to solve problems of chemoselectivity, in order to modify the functional array of the taxanes around the molecule's core. From these efforts, a clearer picture of the role of the various functionalities on the mode of action of the taxanes is emerging. Some issues remain to be resolved, but the next obvious step is to try to design new, easily accessible, scaffolding systems that may hold the essential binding elements found in Taxol |

in the right spatial configuration for

proper binding to microtubules. In addition it is obvious that, among the hundreds of synthetic analogs of Taxol | that have been prepared, some had to have better binding properties or cytotoxicity than Taxol | itself, and this is being reported with increasing frequency. A fuller evaluation of these analogs in vivo is, of course, needed before unrealistic claims are made. Analogs

endowed with better potency or lower toxicity will probably emerge. In addition, since the primary mechanism of resistance to the taxanes appears to be of the mdr type, it seems reasonable to assume that a taxane within a tumor cell will be available for binding with microtubule structures and with the P-glycoprotein (the export p u m p ) in a competitive fashion. While decreased affinity for the promiscuous export p u m p seems hard to engineer, higher affinity for the target (already precedented) may have the overall effect of increasing intracellular drug concentration by shifting the equilibrium in favor of the tubulin-drug complex, and therefore may reduce or suppress resistance

in vitro and perhaps even in vivo. The answers to all these

questions have not emerged yet, but will likely be the subject of future investigations. In C h a p t e r

6, Dr. Joydeep Kant discusses

SAR aspects of the

phenylisoserine side chain. The C-13 side chain is an important element in the binding of Taxol |

to its biological target, and it has become necessary to

devote a whole chapter to this topic in view of the many chiral approaches to phenylisoserines for SAR studies and the m a n y analogs prepared. With baccatins becoming readily available, the easiest modifications that can be made are the ones that incorporate new side chains, and there is no doubt that medicinal chemists will be active in this area for years to come. The issue of how the side chain folds both in solution and at the binding site has piqued the interest of many workers and is also discussed in this chapter.

Finally, in Chapter 7 Prof. Timothy Macdonald and Lisa Landino discuss the mode of action of the taxanes and the mechanisms of resistance. The dynamics of microtubules and the many sophisticated controls for the formation of these interesting and important structures from soluble tubulin are given special emphasis. Learning the precise binding site of Taxol | within its target should help design better analogs, or even attempt

de n o v o

design of Taxol | mimics with completely different structures. In addition, the understanding of how cancer cells become resistant to taxanes may help develop new strategies and modalities in cancer chemotherapy. Research in the Taxol|

area is proceeding at record pace. About half of

what we know about this drug and its analogs, at least measured in terms of number of papers, has been learned in the last two years. An analysis of publications in this area (Figure 1) shows, after the slow 70's, a more rapid phase in the 80's, with

ca.

100 papers/year, followed by an exponential phase

in the 90's coinciding with the clinical development of Taxol |

In 4 years the

number of papers published yearly has increased almost ten-fold, although the relative growth seems to be slowing and perhaps ready to plateau. 1000 900

800 700 600 o

500 400

~ aoo 200 100

0 oO O~

~cO O~

~ o13 O~

r cO O~

~I" oO O~

~ I~0 O~

r4D oO O~

~ o13 O~

~ ~ O~

O~ o13 O~

0 O~ O~

~-O~ O~

04 O~ O~

03 O~ O~

year Figure 1: Publication trend in the taxane area over the last 14 years (searched through Current Contents and Medline)

O~ O~

I surmise therefore that an update of the field in 1995 is especially timely and justifiable. Finally, one w o r d about names: taxol was the name given by its discoverers to the active principle of Taxus brevifolia. The Bristol-Myers Squibb Company, on registration of this c o m p o u n d as its own brand, has made the unusual and perhaps unfortunate choice of registering the trivial name as its own trademark. The c o m p o u n d should then be referred to as Taxol |

Since the generic name has been withdrawn, the company had to

choose a new generic name for the c o m p o u n d , and picked "paclitaxel", certainly not an attractive choice. This has generated confusion in the literature, and in general workers in the field, with the exception of BMS workers, have obvioulsy not paid any attention to the name change, and continue to use the term "taxol" instead of "paclitaxel". In this book, we use the name Taxol |

and paclitaxel, but not the old trivial name. Rhone-Poulenc

Rorer has followed suit, registering the trivial name of their clinical candidate as their o w n trade name (Taxotere |

and baptizing the generic version

"docetaxel". Whatever their names, I am certain that these derivatives will continue to be at the center of the attention in the field of cancer chemotherapeutics, and I hope this book will help workers to advance the field with newer, even more exciting results. A c k n o w l e d g e m e n t s : I wish to thank Dr. Helen Oen (Boehringer Ingelheim, Scientific Information) for keeping me up to date on the copious literature in this area over the last two years, and Mary Feron for extensive retyping of parts of the manuscript. I dedicate this book to my parents, Renato and Wanda Farina.

Vittorio Farina Boehringer Ingelheim Pharmaceuticals 900 Ridgebury Rd Ridgefield CT 06877

Ridgefield, 2/6/95

The Chemistry and Pharmacology of Taxol and its Derivatives V. Farina, editor 9 1995 Elsevier Science B.V. All rights reserved

1 NATURALLY OCCURRING TAXOIDS Giovanni Appendino Dipartimento di Scienza e Tecnologia del Farmaco, via Giuria 9, 10125 Torino, Italy

1.1. I N T R O D U C T I O N

Taxoids (taxane diterpenoids) are a structurally homogeneous class of compounds that occur in two genera of the yew family (Taxus and Austrotaxus). The very limited distribution of taxoids within the plant kingdom is the result of the peculiar taxonomical position of the yew tree, which stands relatively apart from the other seed plants. Furthermore, fossils of ancient yews (Paleotaxus rediviva, T. jurassica, T. grandis) show a close similarity to the modern yews, suggesting a limited evolution through the ages. Part of the remarkable adaptability and evolutionary longevity of the yew tree is presumably related to its complex and peculiar secondary metabolism. Indeed, ancient trees like the yew and the gingko are a storehouse of biologically active compounds, whose complex and unique molecular frameworks give us a glimpse of the biochemical virtuosity of early plant chemistry. The most famous yew (T. baccata L.) is considered a dying-out species. It has become almost extinguished in natural plant communities, and only survives as an important element of green areas (parks, gardens). With the current rate of plant extinction, one may wonder how many taxols and gingkolides will be missed by future generations of plant chemists.

1.2. S Y S T E M A T I C S OF THE YEW T R E E

The yew family ( T a x a c e a e ) has only five g e n e r a ( A m e n t o t a x u s , Austrotaxus, Pseudotaxus, Taxus and Torreya) [1, 2]. Other genera t h a t used to be included in the Taxaceae family (Cephalotaxus,

P o d o c a r p u s ) a r e now

considered part of independent families. Owing to the absence of seed cones, the yew family has sometimes been excluded from the conifer order, raising its taxonomic state to a new order or even class. The systematics of the genus T a x u s are controversial. The yew tree is distributed t h r o u g h o u t the n o r t h e r n hemisphere, and occurs in eight distinct geographical regions. Little, if any, overlap between these enclaves exists, and yews are commonly named from their area of distribution (Table 1). Yews look very much alike, and the presence of only one collective species (T. baccata L.) is often assumed. A classification of this type was proposed by Pilger, who divided the genus into seven subspecies (see Table 1) [1]. However, more recent dendrological work recognized the infraspecific taxa as independent species (e.g. Krfissmann [2], Table 1), and this opinion prevails nowadays. In addition to the n a t u r a l species, two infraspecific hybrids are shown. They originated from cultivations in North America, on breeding the Japanese yew with the European yew (T. x media Rehd.) and with the Canadian yew (T. x hunnewelliana Rehd.). The needles of one cultivar of T. x media Rehd. (T. x media Rehd. cv. Hicksii) contain Taxol| and Taxol-related compounds in amounts comparable to those reported from the bark of the Pacific yew, and might become an i m p o r t a n t and renewable source of Taxol |

[3]. The genus Austrotaxus is monotypic, and its

only species (A. spicata Compt.) is endemic to the New Caledonian rain forest. Confusion exists in the phytochemical literature as to the identity of the Himalayan and the Chinese yew. The former is often referred to as T. baccata L., following Pilger's classification [1] and the Index Kewensis, whereas the Chinese yew, referred to as T. mairei Hu ex Liu, T. chinensis Rehd., T. y u n n a n e n s i s Cheng et L.K. Fu or T. celebica (Warburg) Li., might actually comprise more t h a n one species. As a result of this taxonomic shuffling, one has to source information on a specific yew under several different Latin names. Especially confusing is the fact t h a t the name T. baccata L. has also been applied to some Asian yews. The distinction between the various yews is difficult, and mainly based on three morphological characters: the length of the needles (10-30 mm), the way

they are attached to the twigs (straight or bent), and the shape of the bud scales (crenate and pointed or non crenate and blunt) [1, 2]. In the absence of fruits, the distinction between the yews and some species of Cephalotaxus and Torreya can be difficult even for the best trained eyes. This is evidenced by the name cephalomannine given to a Taxol| analog isolated from a plant identified as Cephalotaxus mannii Hook at the time of collection and chemical analysis [4], but later recognized as a yew species (T. waUichiana Zucc.) [5]. Table 1. The Systematics of the Genus Taxus

Trivial name

Pilger classification [1]

Krtissmanm classification [2]

European yew

T. baccata subsp, eubaccata Pilger

T. baccata L.

Himalayan yew

T. baccata subsp. wallichiana (Zucc.) Pilger

T. wallichiana Zucc.

Chinese yew

T. celebica (Warburg) Li. T. cuspidata Sieb. et Zucc.

Japanese yew

T. baccata subsp, cuspidata (Sieb. et Zucc.) Pilger

Pacific yew

T. baccata subsp, brevifolia (Nutt.) Pilger T. baccata subsp, globosa (Schlechtd.) Pilger

T. brevifolia Nutt.

Florida yew

T. baccata subsp, floridana (Nutt.) Pilger

T. floridana Nutt.

Canadian yew

T. baccata subsp. canadensis (Marsh.) Pilger

T. canadensis Marsh.

Mexican yew

T. globosa Schlechtd.

T. x media Rehd. T. x hunneweUiana Rehd. Many varieties and cultivars of yew have been developed for ornamental purposes. Krtissmann lists 139 of them, giving clues to their identification [2]. No comprehensive survey on the phytochemical pattern of the various yew species, hybrids and cultivars exists. However, some general trends are

l0 emerging, at least at the species level, from the wealth of data published in the last few years (see section 1.10). 1.3. HISTORICAL P E R S P E C T I V E

The first studies that correctly established the constitution of the taxane nucleus appeared in 1963, when three groups (Lythgoe's [6], Nakanishi's [7] and Uyeo's [8]) independently reported their conclusions regarding the carbon skeleton of some Taxus constituents. However, interest in the chemistry of the yew tree dates from the mid-nineteenth century, since a mixture of taxoids was obtained by the German pharmacist Lucas as early as in 1856 [9]. Reading a report by a French veterinarian on the poisonous properties of the yew, Lucas remembered a fact occurred in his own town (Arnstadt) two decades earlier, when a flock of sheep had been placed in a fenced yard landscaped with a few large yew trees. The following day five or six sheep (he could not remember exactly) had died, and he was contacted by a veterinarian, who asked him to analyze the stomach of the dead animals. Poisoning from heavy metals was suspected, but Lucas could not find any evidence for this. Inspection of the yard where the sheep had been kept revealed that the yews had been stripped of leaves as high as the animals could reach. Poisoning from the yew seemed the most plausible explanation for the death of the animals, and the yard's owner had all the yews eradicated from his property. The report by the French veterinarian and his own experience made Lucas suspect the presence of alkaloids in the yew. Many compounds of this type had already been isolated, and some of them were highly poisonous (strichnine, nicotine, coniine). After a laborious extraction, Lucas obtained an amorphous white powder showing basic properties. He named the material taxine, -ine being the ending given at that time to alkaloids. An improved isolation procedure was worked out by Marm~ twenty years later [10]. His preparation gave a crystalline and apparently purer material, but this claim was not substantiated by later workers, who always described taxine as an amorphous powder giving amorphous salts. The first systematic investigation on the biological properties of taxine was carried out by Borchers in 1876 [11]. He recognized the high toxicity of this alkaloid and described its action on the respiratory system and the heart.

ll The structural characterization of taxine was extremely slow. The early studies were unsuccessful, and the first clue came only in 1923, w h e n Winterstein showed that taxine is the ester of a polyalcohol esterified with acetic acid and (L)-~-dimethylamino-~-phenylpropionic acid [12]. Divergent physical constants were reported for taxine (mp 82-124~

[a] D +35-96~ and these early

studies could not dispell the obvious suspicion that taxine was actually a mixture of compounds. A breakthrough came in 1956, a hundred years after the isolation of taxine, when Graf showed t h a t this alkaloid is a mixture of at least seven compounds [13]. G r a f w a s able to obtain three of them in pure form (taxine A, B and C, Figure 1), but the structural elucidation of these compounds was achieved only recently [14-16]. HO AcO'"

I

O

~ ~ sIlI ~

0

O

NMe2

RO 3.1.1 3.1.2

Taxine A R=Ac T a x i n e C R=H

O

.O.

NMe 2

OH 3.1.3 Taxine B Figure 1: The Taxines A different approach was followed by Lythgoe, who discovered t h a t chemical modification of taxine can afford pure compounds. Thus, after acetylation and conversion of the Winterstein esters into cinnamic esters, two products were obtained (5-cinnamoyltriacetyltaxicin I and II), whose constitution was established in 1963 [6], and stereochemistry three years later [17]. The same

12 approach

was

followed

by

Nakanishi

[7]

and

Uyeo

[8].

5-

Cinnamoyltriacetyltaxicin II turned out to be identical to taxinine, a compound obtained in 1925 by Kondo and Takahashi from the needles of the Japanese yew [18]. Taxinine is thus the first natural taxoid to be obtained in pure form and structurally elucidated (Figure 2). The fact t h a t taxine was characterized well after the advent of chromatographic techniques is surprising, since the toxicological relevance of the yew tree has not diminished, and cases of human and animal poisoning are still reported on a regular basis [19]. One has to consider, however, that taxine is unstable, being decomposed by acids and light, and that many of its constituents are prone to isomerization during the purification procedure (see section 1.8). Furthermore, two seminal discoveries shifted the attention of the scientific community towards other constituents of the yew tree. In the late 1960s, interest in taxine was overshadowed by the discovery of the outstanding antitumor properties of Taxol | [20] and by the detection of large amounts of ecdysones in yew tissues [21]. The isolation and structural elucidation of Taxol| reported by Wani and Wall in 1971 [21], was a remarkable accomplishment, because of the low concentration (ca. 0.02% of dry bark weight) and structural complexity of this compound. AcQ

OAc O ,,,,O~jl

0 R 3.1.4 3.1.5

OAc

R=OH (5-Cinnamoyltriacetyltaxicin I) R=H (5-Cinnamoyltriacetyltaxicin II, Taxinine) Figure 2

In those years, systematic studies on the non-alkaloidal constituents of the yew were undertaken by the groups of Nakanishi in Japan and Halsall in England. These groups discovered new members of the taxane group of diterpenoids. Halsall in particular is responsible for the numbering of the taxane skeleton used today, and for the isolation of the baccatins. His entire work was published as a series of six short notes [22-27], and details on the isolation of

13 these important compounds were never reported. In the 1980s, Potier's group in France isolated several new taxanes, including a structural type characterized by a C-12, C-17 oxygen bridge [28], and several analogs of Taxol|

[29]. Other

major achievements by the French group were the first partial synthesis of Taxol| described in 1988 [30], and the discovery of the excellent antitumor properties of Taxotere | a semisynthetic taxane now in advanced clinical study [31]. The partial synthesis of Taxol|

from 10-deacetylbaccatin III, a taxoid

available in relatively large amounts (up to 0.1%) [30] from a renewable source (needles and clippings of several yew species) solved the supply problem and paved the way for the commercialization of Taxol| as a drug. The literature on naturally occurring taxoids was reviewed by Kingston et al. in 1993 [32]. Their work updated a series of previous reviews, summarizing

what was known on the occurrence and the reactivity of taxoids up to March 1992. This work covers relevant literature up to, and including, August 1994.

1.4. REPRESENTATIONS, NUMBERING AND TRIVIAL NAMES Taxoids are compounds having a [9.3.1.03, 8] tricyclopentadecane ring system or closely related skeleta. The bidimensional representation of the taxane skeleton offers the opportunity for a number of drawing modes, but gives no clue as to the actual shape of the molecule (see Figure 10 in chapter 2). Two planar representations are in use (Figure 3): the linear one, by Lythgoe (A) [6], and the angular one, by Miller and Kingston, (B) [32,33]. Both have merits and drawbacks. In both representations, the orientation of the substituents at the tetrahedral ring carbons of rings B and C is represented by the conventional stereochemical symbols (thickened and broken lines) with reference to the a and faces of the molecule. These are defined as in steroids (a=lower face, ~=upper face), observing the molecule with the methyl group at C-8 (C-19) placed in the "northern hemisphere" and pointing toward the observer. The absolute configuration of the natural taxoids is such that, if the molecule is oriented in this way, ring A is to the left and ring C to the right side of the observer. If the taxane ring system is drawn according to Lythgoe (A), ring B is not in its most expanded form, and C-15 is the apex of a reentrant angle. Its substituents must thus be drawn inside ring B. Since reentrant and vertex angles are related by a C2-rotation along an axis passing through the Cn-1 and C n + l atoms, the meaning of thickened and broken lines is reversed, and the actual orientation of

14 a substituent is the opposite of what is intuitively expected. Therefore, the C-16 methyl group, cis to the C-19 methyl and ~ according to the steroid convention, is represented here by a broken line, and the opposite is true for C-17. 18

10

13 / a

9 19(17)

1~.~'(20) r I

"'

2

A

16

6

m(16)

H

B

C

Figure 3: Bidimensional representation of the taxane stereoparent (old methyl numbering in parentheses). In the Miller-Kingston representation (B), ring B is drawn in its most expanded form, and the actual orientation of the gem-dimethyl groups is straightforward from their stereochemical symbols. However, this representation is difficult to draw rigorously, since the perimeter of ring A is too small to contain, in graphical terms, the gem-dimethyl groups. Thus, these are placed into different rings: C-16 (~, thickened line) into ring B, and C-17 (a, broken line) into ring A. Another point of concern is the way the orientation of the substituents on ring A and on the bridgehead carbon C-1 are indicated. Lythgoe considered C-1 as a cyclooctane ring B carbon joined by a three-carbon a-chain (C-12 through C14) to C-11 [17]. The substituent at C-1 (hydrogen of hydroxyl) is thus ~ (cis to the methyl at C-8), but descriptors different from a/~ are needed for the other ring A substituents, since the fragment C-12 through C-14 is considered a t r a n s a n n u l a r bridge and not part of the main ring system. For these substituents Lythgoe used an exo/endo notation [17]. The stereochemical descriptors for the Lythgoe and Miller-Kingston representations are based on the B-C ring system, and the C-1 to C-14 bond should be drawn using a broken line. In practice, the perimeter of the ring system is drawn using a line of normal thickness for this bond as well, and this conventional representation is well established. It must be emphasized, however, that in the current bidimensional representation of taxoids, the stereochemistry at

C-1 is actually not indicated, since only one stereochemical descriptor is

15 employed for the substituents of this stereogenic carbon. This is unambiguous in fused systems, but not in bridged systems. Furthermore, the a/~ notation is applied to taxoids in a rather peculiar way, not fully consistent with the steroid rules. In all n a t u r a l l y occurring taxoids, the non-nuclear C-1 substituent (hydrogen or hydroxyl) is cis to the C-19 methyl, and ~ according to the Lythgoe convention. The most correct and practical representation of the taxane skeleton would be one that considers it as a cyclodecacyclohexane with a methano bridge, and the Chemical Abstract name of taxanes is based on this ring system (Figure 3, C). As with the gem-dimethyl groups of camphor, no stereochemical descriptor would be necessary for C-16 and C-17, which could be written with lines of normal thickness but in different rings (cf. the Miller-Kingston representation [22, 32]), and defined not as a/~, but as syn or anti to a certain element (e.g. the C-19 methyl group). Furthermore, all substituents along the periphery of the ring system would be defined by the same descriptors (a/~), avoiding the cumbersome use of syn / anti for those at C- 12, C- 13 and C- 14. The stereochemistry at C-1 could also be clearly indicated, making the course of some reactions obvious from simple inspection of the bidimensional r e p r e s e n t a t i o n (e.g. the syn allylic epoxidation t h a t established the C-1 functionality in Nicolaou's synthesis of Taxol | [34]). The numbering of the taxane skeleton is based on the IUPAC name of the parent ring system (4,8,12,15,15-pentamethyltricyclo[9.3.1.03,S]pentadecane), and was proposed in 1964 by the leaders of the three major groups working on this class of compounds (Lythgoe, Nakanishi and Uyeo) [35]. In 1969, Halsall proposed a different numbering for the methyl groups [23], similar to the one used in cembranoids and other diterpenoids (tiglianes, daphnanes, ingenanes), where the gem-dimethyl groups are C-16 and C-17. Both systems were used during the following decade, but in 1978 the IUPAC blue book adopted Halsall's numbering [36], which has been the only one used since then. It must be emphasized, however, that in 2(3->20)abeotaxanes (taxine A-type compounds) biogenetic and structural numberings are different (see section 1.6). Many taxanes have been assigned trivial names, derived from the botanical name [baccatins (Figure 4), austrospicatine, brevifoliol] or the geographical location (taiwanxan) of the yew tree where they were first found. A combination of both has also been employed (taxagifine). Many trivial names of taxoids begin with the syllable tax, resulting in confusing proliferation of similar

15 names, often with additional suffix numbers (taxagifines) or letters (taxinines, taxchinins, taxchins, taxuyunnanines). Particularly frustrating is the situation with the taxinines, since only a few scattered letters have been employed (A,B,E,H,J,K,L,M, Figure 5), a situation reminiscent of that of gingkolides (A,B,C,M). The structure of baccatin II is not known. Based on the molecular formula [22], this compound may be 1-hydroxybaccatin I, which generally cooccurs with baccatin I. AcO

OACoAc

AcO

A c O ....

O OH

H O .... R"

v

H

iiii

O

OAc

OAc

HO

4.1.1 R=H, Baccatin I 4.1.2 R=OH, Baccatin II (?)

OBz

OAc

4.1.3 7~-OH, Baccatin III 4.1.4 7(z-OH, Baccatin V OAc " OAc

AcQ

A c O .... O HO

OR

OAc

4 . 1 . 5 R=Ac, Baccatin IV 4.1.6 R=Bz, Baccatin VI 4 . 1 . 7 R=n-Hexanoyl, Baccatin VII

Figure 4: The baccatins Further confusion arises from the fact that also non-taxoidic compounds isolated from the yew (taxicatine) and even synthetic products (taxilan, taxylone) begin with the syllable tax. To complicate matters even more thoroughly, spelling problems exist. Thus, in the English literature, the alkaloidal mixture from the yew has been referred to both as taxin or taxine, but baccatins are taxoids and baccatine is a triterpenoid [37]. Furthermore, baccatin and baccatin I are the same compound [25], but taxinine [7, 8] and taxinine A-M (Figure 5),

17 referred to as taxinin and taxinins A-M by Chemical Abstract, are different compounds. AcO

o

OAc

AcO ~,

OAc ,~ OAc

_

Y

OAc

.... OOinn

OAc

4.1.10 Taxinine B

3.1.5 R=Cinn, Taxinine 4.1.8 R=H, Taxinine A 4.1.9 R=Ac, Taxinine H AcO OAc R

AcO ~,

OAc ~ R

AcO ....

O

....OCi an

"'OR OAc

OAc

4.1.13 R=H, Taxinine K 4.1.14 R=Ac, Taxinine L

4.1.11 R=H, Taxinine E 4.1.12 R=OAc, Taxinine J AcO AcO j / O n

0

" ' " ""

I

Bz

" /OAc

, .... Iss I

OH

OAc

4.1.15 Taxinine M Figure 5: The taxinines

More t h a n one trivial name has been assigned to the same compound, and taxchinin A [38] and 2a-acetoxybrevifoliol [39] are the same compound, as are t a x a c u l t i n [40] a n d taxol D [41]. To avoid this i n t o l e r a b l e confusion, a nomenclature system based on the names of only a few basic taxane structures would be highly desirable. Figures 4 and 5 show the formulas of the baccatins

18 and taxinines. Most of these compounds were structurally elucidated in the sixties and seventies, and their names are rooted in the literature. Whenever possible, plant chemists should try to name new taxoids as derivatives of these compounds. Taxols are baccatin III derivatives esterified at C-13 with phenylisoserines bearing various N-acyl and N-alkyl groups. An alphabet system was proposed by Potier [29], using suffix letters to distinguish between compounds bearing different N-acyl groups (Figure 6). New letters are introduced in alphabetical order whenever a new taxol is isolated [40-42a]. This system allows a rational naming of closely related compounds, and deserves widespread use. Also, it avoids the misleading name cephalomannine for N-debenzoyl-N-tigloyltaxol (now taxol B). Chemical modification within the diterpenoid core can occur naturally at C-7 (epimerization [32, 42c], xylosidation [29]), C-9 (reduction [42b]) and C-10 (deacetylation, oxidation [32, 42c], esterification with ~-hydroxybutyric acid [29]). Overall, twenty-two natural taxols are known to date.

RIN'R20 phil"_

AcO

O OH

0 ....

oH

HO

-

BzO

OAc

4.1.16 RI=BZ, R2=H, Taxol A (Taxol) 4.1.17 Rl=Tigloyl , R2=H , Taxol B (Cephalomannine) 4.1.18 Rl=n-Hexanoyl , R2=H , Taxol C 4.1.19 Rl=n-Hexanoyl, R2= Me, N-Methyltaxol C 4.1.20 Rl=n-Butanoyl , R2=H , Taxol D (Taxacultin) Figure 6: The taxol alphabet 2.5. CHEMODIVERSITY AND STRUCTURAL TYPES 2.5.1 Skeletal types Natural taxoids are rather homogeneous in functional complexity. Indeed, compared to other classes of terpenoids, the structural variations within taxoids

19 are limited, and many compounds only differ in their esterification pattern (e.g. baccatins IV, VI and VII, see Figure 4). The main structural diversity was found within taxanes from the tropical species Austrotaxus spicata Compt. [43, 44], and interesting new structural types might be present in other yews that cross the equatorial line (Indonesian and Malayan yews). However, these plants have not yet been investigated from the botanical or the chemical point of view. Besides taxanes, three other skeletal types of natural taxoids are known (Figure 7), resulting from closure of an extra ring between C-3 and C-11 (3,11cyclotaxanes), or from rearrangement of ring A and ring B [(11(15->1)- and 2(3>20)abeotaxanes, respectively)]. Taxanes are by far the most widespread skeletal types of taxoids, accounting for 96 out of 101 natural taxoids listed in Kingston's review [32, 39], but recent studies have highlighted the importance and widespread distribution of the other minor skeletal types [16, 39]. The four skeletal types of taxoids are exemplified by taxinine [18], brevifoliol [39, 45], taxine A [14], and taxinine K [46], the first compounds of each type to be isolated (Figure 7). Under mild conditions (see section 1.8), certain 13- or 9-oxo-All-taxoids can be turned into 3,11-cyclotaxanes [46, 47], and C-1 hydroxylated All-taxenes can rearrange to 11(15-> 1)abeotaxanes [48]. Taxanes and 2(3->20)abeotaxanes actually have a different biogenesis (see section 1.6). Many other skeletal types have been obtained by radical, cationic or anionic rearrangements of taxanes (see chapter 5), but none of them has so far been encountered in nature. 1.5.2. Functionalization of the terpenoid core Taxanes: The site of main structural variation in the terpenoid core is the C-4/C-20/C-5 moiety. According to its functionalization, taxanes can be divided into five different structural types (Figure 8): 5a-hydroxy-A4,2~ (olefin-type, A), 5a-hydroxy-4~,20-ether type (epoxide-type, B), 4r (oxetane-type, C), 4(z,5a, 20-triol-type (D) and 5~,20-diol-type (E). The tertiary hydroxyl at C-4 is generally esterified, but the secondary hydroxyl group at C-5 and the primary one at C-20 can occur both in free and esterified form. Oxo bridges are always ~, an important observation in regard to the biogenesis of these structural types.

20 AcO ~,

OAc -

0

....0 . ~ ~

Ph

OAc 3.1.5 Taxinine, a taxane OBz OAc " OAc

AcO 11

HO ....

O -

OAc

"r "'.. sssS

s~ 0

OH

OAc

HO 5.1.1 Brevifoliol, a 11(15-> 1)abeotaxane

4.1.13 Taxinine K, a 3,11-cyclotaxane

O NMe 2 ~ , ~ ....0 Ph

AcO . . . . . . . . ..-" AcO

2o

OH

3.1.1 Taxine A, a 2(3->20)abeotaxane Figure 7: Skeletal types of natural taxoids Another i m p o r t a n t point of variation is the oxidation state of C-9 and C13, where both a hydroxy or a keto group can occur (Figures 4 and 5). However, four compounds with unfunctionalized C-13 [23, 49, 50], three compounds with both C-9 and C-13 unfunctionalized [23, 50, 51], and one compound with unfunctionalized C-9 [50] have been described. C-l, C-2, C-7 and C-14 can be oxygenated or not, w h e r e a s C-6 is always unfunctionalized. The t e r t i a r y hydroxyl at C-4 is generally esterified, but the secondary hydroxyl group at C-5 and the p r i m a r y one at C-20 can occur both in free and esterified form.Oxo

21 bridges are always ~, an important observation in regard to the biogenesis of these structural types.

5 '"OH(OCOR)

_ "OH(OCOR)

20 A

B

~

C

"OH(OAc)

'"OAc

1

OH(OAc) D

O OAc(OH)

OAc E

Figure 8:C-4/C-20/C-5 Functionalization types All of the natural taxanes, with the exception of taxagifine and its derivatives, have a double bond at C-11/C-12. This plays an important role in stabilizing the twist-boat conformation of the cyclooctane moiety of the taxanes and in preventing transannular interaction between the substituents at C-3a and C-12a [53]. Indeed, A ll-taxenes are an important example of "hyperstable olefins", where the linear and the angular strain associated with the introduction of a bridgehead double bond are overridden by the decrease in t r a n s a n n u l a r interactions and I-strain caused by the conversion of tetrahedral carbons into trigonal ones [54]. Various combinations of functionalities can occur, as shown in Figures 4 and 5. However, important associations of functional groups exist, possibly due to the presence of "gene cassettes". For example, all oxetane-type taxoids are also oxygenated at C-2 and C-7 (cf. baccatins II-VII, figure 4), whereas all taxoids with a C-13 keto group bear a double bond or an epoxide at C-4/C-20 (cf. taxinines A, B, H, K, L and M, Figure 5).

22 AcO AcO

I

"

OAc

0

0 ....O " j ~ " H

Ph

: OAc 5.2.1

Figure 9: Taxagifine, on oxo-bridged taxane 3,11-Cyclotaxanes: All the non-alkaloidal compounds of this type isolated to date are phototaxicin I and II derivatives [46, 55, 56], and have the same acylation pattern as their corresponding and co-occurring taxicines. However, the taxane corresponding to the 3,11-cyclotaxane pseudoalkaloid spicaledonine (2a-acetoxycomptonine) is unknown as a natural product [44]. AcO

~,

0

OAc

-

"i, "-.. ....OR 2

OAc 5.2.2 RI=OH, R2=Cinn , Triacetyl-5-cinnamoylphototaxicin I 5.2.3 RI=H, R2=Cinn , Triacetyl-5-cinnamoylphototaxicin II 5.2.4 RI=H , R2=COCH(OH)CH(NMe2)Ph , Spicaledonine Figure 10: Some naturally occurring 3,11-cyclotaxanes 11(15->1)Abeotaxane~: All natural compounds of this class (see 5.1.1, Figure 7), bear an oxygen function at C-15 [39], whereas acidic treatment of C-1 hydroxylated taxanes gives mostly rearranged products of this class, but with a C-15/C-16 double bond [48]. Oxygen bridges can form between C-15 and C-10 [57] and between C-15 and C-13 [58], presumably by intramolecular nucleophilic quenching of cations centered at C-15 and C-13, respectively. No 11(15>1)abeotaxanes with a C-13 keto group have been reported.

23

2(3->20)Abeo.taxanes: All compounds of this type are derivatives of taxine A [14] (3.1.1, Figure 7), and only differ in their acylation pattern [16, 59]. 1.5.3. Acylation patterns All hydroxyl groups of taxoids, with the exception of that at C-1, can be found esterified with various acids and aminoacids (Table 2) [60, 61]. As a result, taxoids can be classified as pseudoalkaloidal or non-nitrogenous. The aminoacid can be nicotinic acid or a series of phenylpropanoid ~-amino acids (Winterstein acid, phenylisoserines, variously O- and N-acylated or N-alkylated). Nicotinyl esters of terpenoids are relatively rare as natural products, and occur mainly in the dihydroagarofuran-type sesquiterpenoids from Celestraceae. Phenylpropanoid ~-aminoacids are instead typical building blocks of yew constituents. Nicotinyl residues are found at O(9), and phenylpropanoid aminoacids at 0(5) and O(13). Aminoacids at 0(5) are N-alkylated, and those at O(13) N-acylated [42]. Two exceptions are known: an O(5)-pseudoalkaloid from A. spicata Compt. with a free amino group [44] and the O(13)-pseudoalkaloid N-methyltaxol C, where the aminoacid nitrogen is both alkylated and acylated [40, 42a]. The aminoacid nitrogen of phenylisoserine can be acylated with benzoic, tiglic, capronic (hexanoic) or butyric acid (cf. the taxol A-D series, Figure 6). The non-nitrogenous acids esterifying the hydroxyl groups of the diterpenoid core can be acetic, cinnamic, benzoic, capronic, a-methylbutyric, [5hydroxybutyric or (z-methyl ~-hydroxybutyric acid. Acetyl residues can be found at the tertiary hydroxyl group at C-4 and at all the secondary hydroxyl groups with the exception of C-14. The other acyl residues have instead a more specific location, suggesting the involvement of selective acylases. Interestingly, the acylation pattern of taxanes and ll(15->l)abeotaxanes is generally different, and benzoyl residues have a wider distribution in abeotaxanes, being found not only at C-2, but also at C-7, C-9 and C-10 [39, 58]. Non-enzymatic acyl migrations have been observed between the hydroxyl groups at C-7, C-9 and C-10 [62-64] and between those at C-2 and C-14 [65] (see section 1.8). It is therefore likely that the acylation pattern observed in some taxoids is the result of both enzymatic and non-enzymatic pathways.

24 Table 2. Common Side Chains of Taxoids Structure

Name

Abbreviation

-OCOCH3

Acetate

Ac

-OCO(CH2)4-CH3

Hexanoate

-OCO(CH2)2-CH3 O OH

Butyrate ~-Hydroxybutanoate

0 a-Methylbutanoate

l-oJ

MeBu

a-Methyl-~Hydroxybutanoate

0 Cinnamate

~- 0

v

-Ph

-ok@h 0

Winterstein acid

NN e

ON' -OCOPh 0

l-o

Cinn

Phenylisoserinate Benzoate

Bz

Tiglate

Tigl

Nicotinate

Also, the n a t u r e of the ester group can be affected by non-enzymatic reactions, since Winterstein acid esters are converted into E-cinnamic esters under mild acidic conditions. This conversion can be sometimes useful for the

25 characterization of alkaloidal taxoids. For preparative purposes, Hoffmann elimination after nitrogen quaternization has been used [66], but the more straightforward method is the Cope elimination of the corresponding N-oxides [67]. Treatment with m-chloroperbenzoic acid in THF turns Winterstein acid esters into E-cinnamates. In alkaloidal taxoids, formation of the N-oxides is much faster than double bond epoxidation, and if only a moderate excess of peracid is employed, quantitative yields of the corresponding cinnamates can be obtained [16]. The N-oxides of phenylisoserine derivatives are more stable and can be isolated, as shown by the preparation of the N-oxide of taxine A [16]. 1.5.4. Glycosidation Patterns Taxol| and some analogs have also been isolated in glycosidic form from woody tissues of T. baccata L. (trunk, roots) [29]. In all cases, the residue was Dxylose and the glycosidic bond was at the C-7 hydroxyl group. 1.6. B I O G E N E S I S

1.6.1. Carbocyclic skeleton The taxane skeleton is a terpenoid, and many authors have speculated upon its origin, but very few biosynthetic studies have been carried out. The early suggestion that taxanes are degraded triterpenoids like quassinoids [8] has been dismissed, and the current view is that taxoids are diterpenoids from both the structural and the biogenetic point of view. Lythgoe was the first to propose a reasonable biogenetic derivation for the taxane skeleton [6b]. His scheme (Figure 11) involves the head-to-tail cyclization of E,E,E-geranylgeranyl pyrophosphate (6.1.1) to a C-15 (taxane numbering) macrocyclic cembrene cation (6.1.2). Quenching of the positive charge by the C-11/C-12 double bond and loss of the C-11 proton might afford a verticillane derivative (6.1.4). A transannular cyclization of the 1,5-diene system of this bicyclic intermediate would eventually give the tricyclic taxane skeleton. The transannular cyclization of a 1,5-diene system is a common leitmotiv in the biosynthesis of isoprenoids, and the relationship between verticillenes and taxanes is the same existing in sesquiterpenoids between germacradienes and eudesmanes. The 2(3->20)abeotaxanes could be derived from a modification of this scheme, mediated by double bond isomerization prior to the 1,5-diene cyclization (for the numbering of 2(3->20)abeotaxanes, see section 1.4), and the

25 3,11-cyclotaxane skeleton is probably the result of the photocyclization of suitable taxane precursors. The ll(15->l)abeotaxanes might derive from C-1 hydroxylated taxanes via a Wagner-Meerwein rearrangement, or, alternatively, from 1,15-epoxycembrene derivative 6.1.5, via a transannular epoxide cyclization.

6.1.1 OPP

6.1.5

6.1.4 Verticillene ~ , ~

6.1.2

Q H+

6.1.6

~ H

H

t

H+

H

OH 6.1.9

11( 15-> 1)Abeotaxane

6.1.7 Taxane I

~2 " 6.1.8

HQ

2

6.1 10

o

2(3->20)Abeotaxane

6.1.11

3,11-Cyclotaxane

Figure 11: Biogenesisof the taxoidic skeleta

27 Although this scheme for the derivation of the taxane skeleta seems plausible, it should be noticed that the configuration at C-1 of most cembranes and all verticillanes isolated from plants is different from that of the taxanes [68, 69], and t h a t attempted cyclization of cembrane and verticillane derivatives failed to give any detectable amounts of compounds with the taxane skeleton [7O]. These observations do not disprove Lythgoe's proposal, but may point to a more subtle mechanism. On the other hand, the course of the cyclization of verticilladienes might be steered toward taxane derivatives by a conformational bias induced by the oxygen functions. Indeed, all naturally occurring taxoids are heavily functionalized, with a number of oxygenated sites spanning from three (taxuyunnanine D, 6.1.12 , Figure 12 [50]) to eleven (taxagifine III,6.1.13 [71]), and no simple taxane hydrocarbon has ever been isolated. Furthermore, the only report of the transformation of a verticillane-type diterpenoid into a compound

friedo-verticillane

with a taxane-like skeleton is the one in the trioxygenated clemeolide, 6.1.14 [72]. 0 \\ Aco

....

HO ... O I "..li_L

, ....

H O

p'.,.... H OH

__Ac 6.1.12, Taxuyunnanine D

O OAc

6.1.13, Taxagifine III

O

AcO

OAc " OAc

~

0

OAc

....

%

~

0 //

AcO,,, "OAc OH

6.1.14, Clemeolide

OAc

6.1.15, Taxchin A

Figure 12: Examples of biogenetically related skeleta

28 A survey of the various synthetic procedures employed to assemble the taxane skeleton [73] highlights the importance of radical and anionic processes, but reactions of this type have rarely been employed by Nature for the assembly of terpenoid skeleta. 1.6.2. C-4/C-20/C-5 Functionalization The transannular cyclization of the 1,5-diene system of verticillanes gives a C-4 taxane cation. This might evolve into a 4(20)-double bond, present as such or in vestigial form (epoxide, diol, oxetane) in all but one (taxchin A, 6.1.15 [74]) of the natural taxoids. The further oxidative elaboration of the double bond, following or concomitant to C-5 oxygenation, has been the subject of much speculation, especially with regard to the formation of the oxetane ring (Figure 13). Potier's group has elaborated a synthetic procedure for the transformation of the 4a,5(z,20 triol system (A) into a 4-hydroxy-5~(20)-oxetane (B) [75], and a similar approach was also used by Danishefsky on a model compound [76]. Both schemes are based on an SN2-type displacement of a 5a-leaving group by the C20 hydroxyl moiety. The sequence triol->oxetane is biogenetically plausible and supported not only by the results of studies on model compounds, but also by the occurrence of 4,20,5-trioxygenated taxoids with the correct configuration at C-4 and C-5 [77]. The role of the 4~(20)-epoxides (C) and their synthetic relationship with the 4(20) olefins are instead not obvious. Halsall postulated the conversion of 4~(20)-epoxides into 4a,20-diols and then into oxetanes via anchimerically assisted opening of the epoxide by a C-5 hydroxyl or C-5 ester carbonyl group [26]. This sequence is plausible, but no study reporting a reaction of this type in taxoids or model compounds has appeared. Direct formation of the 5~(20)-oxetane from natural 4~(20)-epoxides via an oxabicyclobutonium ion (D, Figure 13) was proposed by Swindell [89], but studies on model compounds did not support the idea. Furthermore, in all natural taxoids, the 4,20-epoxide is ~ [41], whereas the epoxidation of A4(20)taxenes afforded exclusively the a epoxides, due to the steric effect of the C-8 ~methyl group [41]. Thus, it is not clear whether the natural 4~(20)-epoxides are the precursors of the 5~(20)-oxetanes, or whether they derive instead from the 4(z,20 diols via an alternative ether bridge formation. Another possibility that should not be discounted is the occurrence of a convergent process, involving more than one path, for the formation of the oxetane ring. In all schemes for the

29 oxetane ring formation, the final step is an SN2-type reaction at C-5, where the leaving group may be a phenylpropanoid aminoacid migrating to C-13, a fascinating hypothesis proposed by Potier [79].

"'OH 20 4,20-dihydroxylation

r 4

20,5-ether formatio~/~

5

,s,

',0~ H

m

H

A

r

"•20,4-ether ormation

r

5

~

4

0

_

OH

,5 "OH

s

s SS

F ~~

4

5

| D

Figure 13: Plausible biogenesis of the oxetane ring of taxoids

1.6.3. Phenylpropanoid Aminoacids Many taxoids have the C-5 or the C-13 hydroxyl groups esterified with phenylpropanoid ~-aminoacids. According to the definition of Hegnauer [80], these taxoids are pseudoalkaloids, since they contain nitrogen, but their cyclic carbon skeleton is not derived from an aminoacid. Winterstein acid and N-alkyl and N-acyl phenylisoserines are typical constituents of yew pseudoalkaloids.

30 Their derivation from phenylalanine has been confirmed by feeding experiments [81-84], but exact details of the various steps are not known, nor have the relevant enzymes been characterized. The observation that cinnamic acid is not incorporated into T a x u s ~aminoacids suggests the involvement of a dyotropic rearrangement, catalyzed by an aminomutase, that turns phenylalanine into its corresponding ~-aminoacid (Figure 14) [84]. This step is reminiscent of the phenyllactate-> tropate rearrangement in the biosyntheis of atropine and related alkaloids [85]. The formation of Winterstein acid from phenylalanine takes place with the exclusive loss of the p ro-R C-3 hydrogen [82], an observation that is consistent with the finding that cinnamic acid is not incorporated into yew ~aminoacids [84]. Indeed, the enzyme phenylalanine:ammonia lyase transforms phenylalanine into E-cinnamic acid by removing the C-3 pro-S hydrogen, and the biosynthesis of cinnamic acid and the yew ~-aminoacids are distinct. Hydroxylation at C-2 and N-acylation or N-alkylation would then complete the biosynthesis of the side chain of taxol- and taxine A-type pseudoalkaloids. Feeding experiments have shown that, during the biosynthesis of Taxol| the side chain is not attached in its final form, but as phenylisoserine, whose acylation takes place after C-13 esterification [86]. As discussed in section 1.5, the yew ~-aminoacids generally occur at 0(5) when N-alkylated and at O(13) when N-acylated [42]. Winterstein acid esters are often accompanied by the corresponding cinnamates, and cinnamoyl groups are thus generally found at 0(5). One exception exists, i.e. the ll(15->l)abeotaxane taxchinin B, 6.1.16(Figure 15), where the cinnamate is at O(13) [87]. It has been suggested t h a t 0(5)- and O(13)-esters are related by the intramolecular migration of the aminoacid side chain from 0(5) to O(13) [79]. Although no experimental evidence for a reaction of this type is known, inspection of models shows the plausibility of this hypothesis, since the taxane skeleton has an inverted cup shape, and the oxygen function at C-13 and C-5 are in close spatial proximity. Doubts on this hypothesis were cast by feeding experiments, since labeled baccatin III could be incorporated into Taxol | even though its functionalization pattern precludes O(5)->O(13)-transesterification [86]. If a simple biogenetic relationship between 0(5) N-alkylated and O(13)-Nacylated alkaloids does exist, its manipulation could allow one to switch the metabolism of yew needles from the abundant (1%) N-alkylated esters like taxine B to the much rarer (ca. 0.004-0.01%) N-acylated esters like Taxol|

31 phJ~1

CO 2H mutase NH2 r p hH ' ~ " v - -CO2 NH 2

(L)-Phenylalanine

N-Methylation = Winterstein acid

1. Hydroxylation 2. Esterificationwith baccatin(ROH) _NH2 _NHBz p h j ~ z / _ CO2R gzCoA ph~:~,~,_ CO2 R _

OH Phenylisoserine

OH Taxol

Figure 14: Biosynthesis of Taxus ~-aminoacids and of Taxol|

BzO

_OACoAc

CinnO .... O HO

OAc

6.1.16 Figure 15: The structure of Taxchinin B

Minute amounts of taxoids are also produced by Taxomyces andreanae, a fungal endophyte of the Pacific yew [88], whose ability to synthesize taxanes is a remarkable example of horizontal transfer of genetic material [89]. The fungal and the plant biosynthesis of Taxol|

could be distinguished with labeled

precursors, since the aminoacid leucine is a precursor of Taxol| in T. brevifolia Nutt. but not in Taxomyces andreanae, where formation of ~-hydroxy-~methylglutaryl CoA (and thus isopentenyl pyrophosphate) from this aminoacid is negligible compared with its formation from acetate [88]. Further details on the fungal biosynthesis of Taxol| are not known. 1.7. C H E M I C A L R E A C T I V I T Y OF T A X O I D S

Taxoids are relatively unstable compounds, being sensitive to acidic, basic and oxidizing conditions. Furthermore, the inverted cup shape of these skeleta

32 and the high density of functional groups make a variety of intramolecular reactions

possible.

We briefly

summarize

here

the

major

types

of

transformations that are somewhat general for taxoids. For a thorough review on the chemistry of

Taxol|

and specifically the synthesis of analogs for

Structure-Activity Relationship (SAR) studies, the reader is referred to chapter 5 and 6. 1.7.1. Skeletal rearrangements Modifications of the carbon connectivity of all three rings of the taxane system have been reported. Most of these rearrangements have been observed in baccatin III derivatives, and are discussed in chapter 5. The focus here is on on rearrangements observed in other structural types of taxoids. Ring A rearrangements: Formation of a cationic species at C-1 triggers the Wagner-Meerwein rearrangement of ring A, resulting in contraction of this ring and formation of a tertiary cationic species at C-15 (Figure 16) [39, 48]. The latter can eliminate an a-proton (7.1.3) or be quenched by a nearby hydroxyl (7.1.4) or a water molecule (7.1.5) [57]. The formation of a cationic species at C13 can trigger two different rearrangements of ring A, depending on the substituent present at C-1 (Figure 17). In C-1 hydroxylated taxanes, the C-1/C15 bond fragments, generating a hydroxylated cation at C-1 and eventually affording cyclodecene derivative 7.1.8 [90]. When a hydrogen is present at C-l, the C-1/C-15 bond fragments in a different way, generating ring-contracted C-15 cation 7.1.10 [22]. Contraction of ring A was observed during the irradiation of certain taxinine derivatives (Figure 18) [91]. In these compounds, the usual photochemical reactivity of 13-oxoA4(2O),ll-taxoids (hydrogen migration from C-3 to C-12 and bond formation between C-3 and C-11) was shut down by the saturation of the 4(20)-double bond or by the formation of the C-9/C-10 acetonide (as in 7.1.14, Figure 18), and the formation of cyclopropanated derivatives 7.1.17 was observed instead, via C-C bond migration between C-1 and C-12. Removal of allylic activation for the C-3 hydrogen, introduction of steric constraints due to the presence of the acetonide methyls, or a longer C-4 to C-11 distance may be responsible for this behavior.

33 HO 1

HO"'

7.1.1

HO

HO ....

7.1.2

Figure 16: Cationic rearrangements of ring A: C-1 trigger T r a n s a n n u l a r cyclizations:

Reactions of this type have been observed under

radical and photochemical conditions. The radical cyclization of baccatin III derivatives is discussed in chapter 5. The irradiation of taxinine-type compounds causes an unusual photochemical reaction, involving hydrogen transfer from C-3 to C-12, the enone (z-carbon (7.1.13, Figure 18). In reactions of this type, hydrogen transfer to the enone [~-carbon or to the ketone carbonyl is generally observed. This r e m a r k a b l e cyclization was discovered by Nakanishi [46], and proceeds via a ~,~* triplet or a C-11/C-12 diradicaloid [89]. The inverted-cup shape of the taxane skeleton makes the hydrogen transfer to the C-11 or to the enone oxygen impossible, whereas H-3 and C-12 are spatially dose and can form a bond.

34

0

7.1.6

7.1.7

7.1.8

7.1.9

7.1.10

7.1.11

H+

Figure 17" Cationic rearrangements of ring A: C-13 trigger Ring B rearrangements" The rearrangement of taxinine to anhydrotaxininol (Figure 19) holds a venerable position in taxoid chemistry, since it was discovered as early as 1931 [92], and represents the first skeletal rearrangement observed in this class of compounds. The rearrangement takes place in a basic madium, v i a a vinylogous retro-aldol fragmentation, followed by aldol condensation between the C-14 enolate and the C-9 formyl, and by SN2-type displacement of the C-2 acetate by the C-14 enolate. The stereochemistry of anhydrotaxininol is not known. 1.7.2. Functional group modifications Hydroxyl groups: The very low reactivity of the 5a-hydroxyl group toward acylation and silylation seems general in all skeletal types. In reactions of this type, the 13-hydroxyl generally shows a lower reactivity than the C-2, C-7 and C9 hydroxyls, and in oxetane-type compounds this is enhanced by the formation of hydrogen bonding with the C-4 ester carbonyl [30]. The relative reactivity of the other oxygen functions depends on the functionalization pattern, and no general rule can be given as to their relative reactivity in esterification or hydrolysis reactions. Furthermore, the reactivity order of the various hydroxyl groups in oxidation reaction does not parallel that observed in acylation and silylation reactions.

3.5

AcO .~

AcO

~'

:~

hv

0

=

0

H-3

, %

OH : OAc

Cinn

3.1.4

H.I AcO~ .,~ O

OCinn migr.

OIH'~_. H ~ OAc 7.1.12

OAc F;~,

AcO

"" " ~ ~ ~ =L_I..., 9 .&P %

OIH"~__ - I OAc

C-3/C-ll bond

OCinn

formation

?Ac

H ....

~ O

",~, "-.-

% "

,,

"OCinn

OAc

7.1.13

5.2.2

oXo .-~

oXo ~ .~ "OAc

H

OAc

Ac

:

OAc 0

0

0

C-l, C-12bond=,O~

~

migration

J, bondform.: O [1 "c~

: 7.1.16

OAc

C-1,C-11

0

X

"OAc

II ~Ac

7.1.17

OAc

Figure 18: Photochemistryofthe 13-oxo-All-enone systemin taxoids

36 AcO

~OAc

HO

(OH

/_,J O

KOH =

O

"OCinn

.... H

OAc

"OH

:

OAc 7.1.18

3.1.5

O I

O Z

"-"~ 0

O I ~

H 7.1.19

OAc O

9

0

H

-

OH

"OH

--7.1.20

OH

0

"OH

OH

OH

%'O

7.1.21, Anhydrotaxininol Figure 19: The rearrangement of Taxinine to Anhydrotaxininol Thus, the allylic 13-hydroxyl, which is difficult to acylate, can be oxidized very easily even with reagents not normally used for the oxidation of alcohols to ketones (MCPBA, OsO4, DDQ, NBS) [93]. The oxidation of the side-chain hydroxyl of phenylisoserine taxane esters leads to a-ketoesters that are stable in the Taxol |

(N-acylation)

series

[94], but are easily degraded to the

corresponding enones, probably via a cyclic mechanism, in the taxine A (Nalkylation) series [16]. Vicinal hydroxyls (C-9/C-10; C-1/C-2 or C-1/C-14) can be protected as cyclic acetals [6b], carbonates [95], or orthoesters [96]. The C-l, C-2 carbonate served as a benzoate precursor in Holton's [97] and Nicolaou's [34] total syntheses of Taxol |

37 Keto groups: In natural taxoids, keto groups occur at C-9, C-10 and C-13. The C9 carbonyl is sterically hindered and unreactive toward reducing agents and other nucleophiles. The C-13 and C-10 keto groups do not react with hydrazinetype reagents [6b], but can be reduced with borane [98] or hydrides [34]. Formation of only the a-alcohol at C-13 was reported in a baccatin III derivative (Figure 20 [34]), but closely related compounds gave mixtures of both epimers as well as the product of overreduction [16, 75]. The unusual electronic structure of All-13-oxo and/or 9-oxo-taxanes is discussed in section 2.3. It is worth noting t h a t the regioselectivity of the reduction of 13-oxo-All-taxanes is rather capricious, and sometimes opposite to what expected. The formation of taxininol (7.2.1, Figure 20) upon t r e a t m e n t of taxinine with lithium aluminum hydride involves a remarkable conjugate reduction of the enone system to a ketone [99]. Furthermore, the 13-oxo group of taxinine-type compounds is totally unreactive under conditions in which baccatin-type compounds are reduced [59]. Double bonds: Reactions of the 4(20)-double bond of taxanes yields predictable products. Thus, this double bond is cleaved by ozone [6b], whereas catalytic hydrogenation [6b], osmylation [75], and epoxidation [41] occur with exclusive attack from the less hindered a-face. The catalytic hydrogenation is plagued by the hydrogenolysis of the C-5 oxygen function, especially in polar solvents [100]. On account of a more caged and h i n d e r e d structure, A4,10-3,11-cyclotaxanes and their hydrogenolysis products are inert toward hydrogenation [100]. The C-11/C-12 double bond is quite unreactive toward electrophilic and nucleophilic addition as well as toward catalytic hydrogenation. Some reactions have occasionally been described, but appear to be associated with a specific functionalization pattern of the taxane skeleton [41]. Thus, the epoxidation of some A4(20),ll-taxadienes occurred preferentially at the endocyclic double bond [41], but in closely related compounds where the hydroxyl groups allylic to the l 1-double bond are acylated, peracid attack occurred mainly at the exocyclic double bond (7.2.7 v s . 7.2.8, Figure 21) [41], whereas under these conditions baccatin III derivatives were oxidized to the corresponding 13-enones (see 7.2.10, Figure 21) [16].

38 1.8. I S O L A T I O N ARTIFACTS

PROCEDURES,

ANALYSIS

AND

EXTRACTION

Two taxoids (Taxol| and 10-deacetylbaccatin) are commercially available. Their isolation from various plant parts has been thoroughly investigated and optimized, but details have not been disclosed for obvious proprietary reasons. AcO ~,

OAc .:

HO

LiA1H4, THF H

:

"OCinn

~ 7

~ ..,

ref. 99 H

3.1.5, Taxinine

OH

OH

OAc

AcO

OH

7.2.1, Taxininol

O OTES

AcO

O~

O OTES

NaBH4, MeOH, 84%~HO .... O HO

: OBz

ref. 34

O

OAc

HO OBz

7.2.2

HO

OAc 7.2.3

20TES

,,

HO ~,

0 / / OTES

% P

O

NaBH4, MeOH, 34% HO" _-

O HO

: OBz 7.2.4

OAc

ref. 16

""

O

OBz 7.2.5

Figure 20: Hydride reductions of the 13-oxo-A11-taxenesystem On a laboratory scale, early studies on the yew focused on the basic alkaloidal constituents, and used aqueous acids for the extraction of the plant material [66]. Neutral and polar solvents (CH2C12, CHC13, methanol, ethanol,

39 acetone) are used nowadays for the extraction of the whole taxoidic fraction. Nonpolar solvents (i.e. petroleum ether) have occasionally been used for the selective extraction of relatively nonpolar taxoids, like the baccatins from the heartwood of T. baccata L. [101]. Extracts from the needles are generally purified by partition between aqueous methanol and petroleum ether. In this way, part of the pigments, i.e. simple phenolics like 3,5-dimethoxyphenol as well as lipids are removed.

RO

OR

OR

\

RO ....

MCPBA _ "O R

H

RO ~, /

RO,,,,~~,.

OR " OR *

CH2C12,

H

ref. 41

0

"O R

7.2.7 7.2.6

+

RO ,

R

7.2.7

H Ac

traces major 80% 20%

O

OR J,

"

OR

7.2.8

Ro

,

"oR 7.2.8

,,,', .o

HO ~,

O

HO

/70_H

0

MCPBA

0

_

O

OBz

7.2.9 R=H 7.2.10 R=OH

CH2CI2, ref. 16

O

OBz 7.2.11 R=H (69%) 7.2.12 R=OH (62%)

Figure 21: Reactions of All-taxenes with peracids

An alternative technique to remove these compounds is the treatment of a 1:1 water-ethanol solution of the extract with lead(II) acetate. A procedure of this type can also be applied to seed extracts for the removal of the abundant lipid

40 fraction [102]. The purified extracts are then separated using conventional chromatographic techniques, open column chromatography on SiO2 still being the most extensively used one. HPLC, both on normal and reverse phase, can be used for f u r t h e r purification of the c h r o m a t o g r a p h i c fractions. More sophisticated techniques (high speed planetary countercurrent chromatography) have also been used [103]. Most taxoids are crystalline compounds, and crystallization is very useful as a purification method. Unfortunately, mixed crystals can sometimes form, especially with taxols [16]. The qualitative and quantitative analysis of yew extracts is routinely carried out by HPLC on reverse phase columns (phenyl, cyano or C-18) [3]. Since the polarity of taxoids varies considerably and the extracts can contain up to 5060 different taxoids in detectable amounts, no universal solvent for the resolution of these mixtures exists. The analysis generally focuses on taxoids having a defined range of polarity (e.g. taxols, baccatin III and deacetylbaccatin III), and selectivity is achieved using UV detection, thus avoiding interference from non UV-active taxoids (brevifoliol, baccatin IV). The speed of the analysis is an important parameter when a large number of samples have to be analyzed, and MS/MS techniques for the detection of Taxol| requiring less t h a n five minutes per sample have been developed [104]. Extensive surveys on taxoid distribution in yew extracts must have been carried out by companies that commercialize taxoids (Indena, Hauser), but these results have not been published for proprietary reasons. This, and the difficulty of obtaining many taxoid standards, is responsible for the paucity of studies devoted to the study of taxoids other t h a n taxols and taxol-equivalent compounds (baccatin III and its 10-deacetyl derivative) in yew extracts. Methods for the q u a n t i t a t i v e analysis of taxoids in plant m a t e r i a l s using mass spectroscopy (FAB, MALD, spray ionization and MS/MS techniques) [104], or immunochemical methods [105] have also been reported. The major problem plaguing the isolation of taxoids is the formation of artifacts, since several reactions of taxanes are possible under conditions similar to those encountered during the preparation of plant extracts and their separation. Acyl .migrat..i.on: Reversible acyl migrations involving the hydroxyl groups at C-7, C-9 and C-10 have been documented in CDC13 solution (Figure 22) [62-64]. In spite of the topological similarity, no reaction of this type was observed in partially deacylated baccatin VI and abeobaccatin VI derivatives [39], suggesting t h a t the a c t i v a t i o n energy for the acyl m i g r a t i o n d e p e n d s on the

41 functionalization pattern of the taxoidic core. However, it should be noted that CDC13 used for NMR spectroscopy does not contain soluble stabilizers, and formation of HC1 may have been responsible for the observed migrations. Indeed, solutions of monoacylated cinnamoyl taxicins in stabilized chloroform are stable for weeks, and this solvent could even be used for their separation [62].

AcO

OH

O ==~

OH

.~ n ,0' ' NMe2 O / u0 ' v ~ ~,;OCin I1 Ph 16 . -NHMe2 ()H ref.

-

HO

AcO

(3H

"-

3.1.3

HO

8.1.1

~OAc

O

~l . HO OH 8.1.2

OAc

HO Q

NMe2

./91,,,O,..Jl,,.~ph

_NHMe20

OH

"OCin

8.1.3

Figure 22: Isomerization/degradation reactions observed in CDC13solution Loss of Dimethylamine from Winterstein Acid Esters: It was recognized very early that taxine is unstable and loses activity upon storage, presumably as a consequence of its loss of dimethylamine and formation of the corresponding cinnamates (Figure 22). This was observed, along with O(10)->O(9) acyl migration, with CDC13 solutions of taxine B [16]. The derivation of cinnamic esters of taxoids from their corresponding Winterstein acid esters seems highly plausible, although it is not clear to what extent this derivation may be biogenetic or simply chemical. At room temperature, prolonged (months) contact with chloroform and silica gel is required for elimination [43], and it may well be that the reaction is also under enzymatic control.

42 For unobvious reasons, Winterstein acid esters with an acetoxyl at C-13 are more stable than those with a keto group at this position, like taxine B [106]. C-7 Epimerization: In taxoids bearing a keto group at C-9, the C-7 ~-hydroxyl can reversibly epimerize via a retro-aldol/aldol mechanism (Figure 23). The reaction has been reported to occur under mildly basic or acidic conditions (basic alumina or silica gel) [57] and even on concentration of HPLC chromatographic fractions [107]. In the a-epimer, the hydroxyl group is pseudoaxial, and this arrangement is stabilized by the formation of a hydrogen bond with the C-4 acetate carbonyl group [108].

H

~HO,,,

Ho

_ HO

-Bz O 7.2.9

,__Y,.;o OAc

L/H_-Y".,J ~ OAc 8.1.6

HO"- r ' HO

O OAc OBz 8.1.7

Figure 23: Retroaldol equilibration of baccatin derivatives [57] Ring A Contraction: In acidic medium, C-1 hydroxylated taxanes can rearrange to the corresponding ll(15->l)abeo derivatives (Figure 16). A mixture of C-15 hydroxy and A15,16-derivatives is formed, in a ratio depending on the reaction conditions [48]. However, all ll(15->l)abeotaxanes isolated to date have a C-15 oxygen function, and their dehydration products have never been detected in yew extracts [39]. Furthermore, the acylation pattern of l l(15->l)abeotaxanes and taxanes is generally different [59]. Thus, baccatin VI is widespread, but its corresponding ll(15->l)abeotaxane (abeobaccatin VI) is unknown as a natural product, since only deacetylated [59] or isomeric (O(10) vs. 0(2) benzoate [39]) derivatives have been isolated. It is therefore likely that 11(15-> 1)abeotaxanes are natural constituents of the yew. More details about this rearrangement are discussed in chapter 5. Photocyclization: Taxinine-type compounds are converted by UV light to their corresponding 3,11-cyclotaxanes (Figure 18) [46]. The reaction is often quantitative, and is accompanied by the E->Z photoisomerization of the cinnamoyl residues [55], and by the reductive deamination of Winterstein acid

43 residues to the corresponding ~-phenylpropionates [100]. The fact that no taxoid with a Z-cinnamoyl residue has ever been isolated points to an enzymatic origin for 3,11-cyclotaxanes. Further details on the photocyclization reaction of taxanes can be found in chapter 5. Reactions During Plant Drying and Storage:

Taxoid fractions obtained from

fresh and dried plant samples can show remarkable differences [16], and the Taxol | content of the needles decreases substantially during drying and storage [109]. The nature of the reactions responsible for this is presently unknown. 1.9. BIOLOGICAL A C T M T Y Interest in the biological activity of taxoids has centered around the tubulin-mediated antitumor activity of Taxol | (see chapter 7 for details). However, cytotoxicity has been reported also for taxoids structurally unrelated to taxols, and yew extracts display various activities, some of which may be due to taxoids and may be of relevance for medicine or toxicology. 1.9.1. Non Tubulin-Mediated Toxicity Taxol | was initially isolated because of his remarkable cytotoxicity to KB cells [20], but yew extracts also contain less cytotoxic compounds, both of taxoidic and non-taxoidic structure (lignans). In particular, some non-alkaloidal taxoids can inhibit DNA replication and protein synthesis in tumor cells [110]. The mechanism for this activity is presently unknown, and only a limited number of compounds have been tested in these systems. The most active one was a A4(2~ [110], but the significance of this finding is difficult to assess without additional information. 1.9.2. Cardiotoxicity The yew has a remarkable history as a poison [111], and the plant is still relevant today in human and veterinarian toxicology. The toxicity of the yew has been ascribed to taxine, whose administration can reproduce the cardiac and respiratory disturbances typical of yew poisoning [112]. Taxine is a heart poison that seems to act on ion channels, blocking sodium and calcium currents [113]. The mechanism of this activity is not known at the molecular level. The study of taxine has been hampered by the instability and the complexity of this alkaloidal mixture. Its LD50 is known only

44 approximately, since no established criteria of purity exist, and the composition depends on the method of preparation and the yew species from which it derives [112]. F u r t h e r m o r e , m a n y studies were done on soluble taxine salts (hydrochloride, sulfate), whose purity and stability may have been different from those of crude taxine. Only one study has been carried out on purified constituents (taxine A and B), showing that taxine B is toxic (LD50 4.5 mg/Kg in mice by intravenous administration), whereas comparable amounts of taxine A are not [114]. Taxine B is the major (20-30%) component of taxine, and should also represent its poisonous constituent. Other components of taxine have been reported recently, but they were isolated in insufficient amount for a detailed in vivo investigation of their cardiotoxicity [106]. Taxol| shows only modest cardiotoxicity [115], but the presence of alkaloidal impurities, and in particular of taxine B, may be clinically relevant, since Taxol | is administered at relatively high doses, and taxine B has been reported to give cardiac anomalies at doses as low as 0.5 mg/Kg [114]. No analytical method for the characterization of taxine has been reported to date. 1.9.3. Other Activities Yew extracts have hormonal activity in insects and mammals. Indeed, the needles of the European yew are a good source of the moulting hormone ecdysones [21]. Block of ovulation and anti-implantation activity have been reported in rabbits and albino rats [116, 117]. Several studies have evidenced a sedative activity in yew extracts [118], and their cosmetic use is also discussed in the patent literature [119]. Apart from the ecdysones, the compounds responsible for these activities have not been identified, but the sedative activity may be due to dimeric flavonoids and not to taxoids. Amentoflavone binds to the BDZ site of the GABA receptor [120], and this compound occurs, along with several other derivatives, in the yew [121]. Among the phenolic constituents of the yew, the phenylbutanoid glucoside rhododendrin also shows hepatoprotective activity [122]. The severe allergic reactions reported upon administration of Taxol| have been ascribed to the vehicle Cremophore EL. However, an almost fatal allergic reaction was reported in a child who had chewed a few needles of yew [123], suggesting that the plant may contain powerful allergens. Contact dermatitis from yew wood has also been reported [124].

45 1.10. TAXONOMICAL R E L E V A N C E A N D RAISON D'ETRE

The distribution of taxoids within the Taxaceae family has taxonomical value, allowing a clearcut distinction between morphologically related genera. Taxoids are so far the only non-steroidal (triterpene) isoprenoids isolated from the genera Taxus and Austrotaxus, and this sets them apart from the genus Torreya, which contains labdane diterpenoids and furanosesquiterpenoids. Curiously, the seeds of some torreyas are edible [125], provided that the aril is removed, whereas the fleshy aril surrounding the seed is the only part of the yew that lacks taxoids and can be eaten. Comparison of the taxoid pattern within the genus Taxus might also help in unraveling the taxonomical relationship between the various species, varieties and cultivars of the yew. However, as usual with secondary metabolites, the production of taxoids is under a complex control mechanism, including genetic and epigenetic factors (environment, season, cultivation practices, plant part). As a result, clearcut conclusions are difficult to draw. The longevity of the yew tree, the presence of sexually distinct plants and the ease of hybridization are additional complications. The greater infraspecific variation of the taxoid pattern regards the basic alkaloids. Indeed, it was recognized very early that taxine from various yew species is different, and that some yews (e.g.T. brevifolia Nutt.) contain only small amounts of it [126]. Hegnauer suggested that the smallest amounts of alkaloids are contained in the species t h a t lacks cyanogenetic glycosides [127], but this correlation has not been investigated further. Some general trends are also emerging in relation to other types of taxoids. Thus, in the European yew, taxicin-type compounds are generally hydroxylated at C-l, whereas in the Japanese yew compounds of this type are generally unfunctionalized at C-1. Furthermore, C-14 hydroxylation seems typical of taxoids from the Himalayan and the Chinese yew, but 2(3->20) abeotaxanes have so far been detected only in the European yew. The production and storage of secondary metabolites is probably the most i m p o r t a n t s t r a t e g y adopted by plants to defend t h e m s e l v e s a g a i n s t microorganisms, herbivores and other plants. Very little is known about the ecophysiological role of taxoids, but the observation that the yew is poisonous to most vertebrates and insects suggests that its secondary metabolites have evolved as part of a defense strategy.

46 Furthermore, the various yew species produce not just a single taxoid, but complex mixtures of a limited amount of major metabolites and a larger number of minor derivatives, as expected for compounds designed to confer a broadspectrum resistance against predators. Thus, the poisonous properties of taxine and the anticancer activity of Taxol | might not be the result of an accidental affinity of these compounds for receptor sites on animal proteins, but may be part of a more subtle strategy whose exact details are elusive. The production of secondary metabolites is believed to develop during evolution by natural selection, and the long evolutionary life of the yew tree indicates that associations seen today may not be those in which the chemical interaction originally evolved. Taxoids may well be important and multitask "fitness factors" for the yew tree, but the plant must also have evolved strategies to avoid autotoxicity, since Taxol| interacts with a cellular structure (the microtubule) present in all eucaryotic cells, and plant tubulin is sensitive to this drug. As the age of molecular medicine dawns, plants represent a still largely untapped source of chemodiversity. The yew tree has provided us with Taxol | but other drugs or pharmaceutical leads may emerge in the future among the unique and fascinating secondary metabolites of this plant.

Acknowledgements I am very grateful to my wife Enrica for her patience and understanding during the time I dedicated to this chapter and not to her. I t h a n k Prof. P. Gariboldi (University of Camerino), Dr. B. Gabetta and Dr. E. Bombardelli (Indena, Milano) for their useful suggestions and comments. I am grateful to all members of my research group for showing me every day that scientists are happy people, and that the essence of science is independent thinking and hard work, not equipment.

REFERENCES .

2.

Pilger, R. Mitt. Deutsch. Dendrol. Ges. 1916, 2~ 1. Krfissmann, G. Handbuch der Nadelgeh6lze (1983), Paul Parey Verlag; Berlin und Hamburg, p.317.

47 .

,

.

.

.

.

.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23.

Witherup, K.M.; Look, S.A.; Stasko, M.W.; Ghiorzi, T.J.; Muschik, G.M.; Cragg, G.M.J. Nat. Prod. 1990, 53, 1249. Powell, R.G.; Miller, R.W.; Smith Jr., C.R.J. Chem. Soc., Chem. Commun. 1979, 102. Miller, R.W.; Powell, R.G.; Smith Jr., C.R.; Arnold, E.; Clardy, J. J. Org. Chem. 198], 46, 1469. (a) Eyre, D.H.; Harrison, J.W.; Scrowston, R.M.; Lythgoe, B. Proc. Chem. Soc. 1963, 271; (b) Harrison, J.W.; Scrowston, R.M.; Lythgoe, B. J. Chem. Soc. (C) 1966, 1933. Kurono, M.; Nakadaira, Y.; Onuma, S.; Sasaki, K.; Nakanishi, K. Tetrahedron Lett. 1963, 2153. Ueda, K.; Uyeo, S.; Yamamoto, Y.; Maki, Y. Tetrahedron Lett. 1963, 2167. Lucas, H. Arch. Pharm. 1856, 85, 145. Marm~, H.W. Bull. Soc. Chim. Fr. 1876,26, 417. Borchers, R. ExperimenteUe Untersuchungen i~ber die Wirkung und Vorkommen des Taxins, GSttingen, 1876. Winterstein, E.; Guyer, A. Z. Phys. Chem. 1923, 128, 175. Graf, E. Angew. Chem. 1956, 68, 249. Graf, E.; Kirfel, A.; Wolff, G.-J.; Breitmaier, E. Liebigs Ann. Chem. 1982, 376. Ettouati, L.; Ahond, A.; Poupat, C.; Potier J. Nat. Prod. 1991,54, 1455. Appendino, G., unpublished results. Eyre, D.H.; Harrison, J.W.; Lythgoe, B. J. Chem. Soc. (C) 1967, 452. Kondo, H.; Takahashi, T. Yakugaku Zasshi 1925, 861. Van Ingen, G.; Visser, R.; Peltenburg, H.; Van der Ark, A.M.; Voortman, M. For. Sci. Int. 1992, 56, 81. Wani, M.C.; Taylor, H.L.; Wall, M.E.; Coggon, P.; McPhail, A.T.J. Am. Chem. Soc. 1971, 93, 2325. Imai, S.; Fujioka, S.; Nakanishi, K.; Koreda, T.; Kurokawa, T. Steroids 1967, 10, 557. Chan, W.R.; Halsall, T.G.; Hornby, G.M.; Oxford, A.W.; Sabel, W.; Bjamer, K.; Ferguson, G.; Monteath Robertson, J. J. Chem. Soc., Chem. Commun. 1966, 923. Della Casa de Marcano, D.P.; Halsall, T.G.J. Chem. Soc., Chem. Commun. 1969, 1282.

48 24. 25. 26. 27. 28. 29. 30. 31. 32.

33. 34.

35. 36. 37. 38. 39. 40.

Della Casa de Marcano, D.P.; Halsall, T.G.J. Chem. Soc., Chem. Commun. 1970, 216. Della Casa de Marcano, D.P.; Halsall, T.G.J. Chem. Soc., Chem. Commun. 1970, 1381. Della Casa de Marcano, D.P.; Halsall, T.G.; Castellano, E.; Hodder, O.J.R.J. Chem. Soc., Chem. Commun. 1970, 1382. Della Casa de Marcano, D.P.; Halsall, T.G.J. Chem. Soc., Chem. Commun. 1975, 365. Chauvi~re, G.; Gu~nard, D.; Pascard, C.; Picot, F.; Potier, P.; Prang~, T. J. Chem. Soc, Chem. Commun. 1982, 495. S~nilh, V.; Blechert, S.; Colin, M.; Gu~nard, D.; Picot, F.; Potier, P.; Varenne, P. J. Nat. Prod. 1984,47, 131. Denis, J.-N.; Greene, A.E.; Gu~nard, D.; Gu~ritte-Voegelein, F.; Mangatal, L.; Potier, P. J. Am. Chem. Soc. 1988,110, 5917. Review: Gu~nard, D.; Gu~ritte-Voegelein, F.; Potier, P. Acc. Chem. Res. 1993, 26, 160. Kingston, D.G.I.; Molinero, A.A.; Rimoldi, J.M. in Progress in the Chemistry of Organic Natural Products: Herz, W.; Kirby, G.W.; Moore, R.E.; Steglich, W.; Tamm, Ch., Eds.; Springer-Verlag; Vienna, 1993; vol.61, p.1.; also: Blechert, S.; Gu~nard, D. in The Alkaloids; Brossi, A. Ed., Academic Press ; New York, 1990; vol. 39, p. 195. Miller, R.W.J. Nat. Prod. 1980, 43,425. Nicolaou, K.C.; Yang, Z.; Liu, J.J.; Ueno, H.; Nantermet, P.G.; Gut, R.K.; Claiborne, C.F.; Renaud, J.; Couladouros, E.A.; Paulvannan, K.; Sorensen, E.J. Nature 1994,367, 630. Lythgoe, B.; Nakanishi, K.; Uyeo, S. Proc. Chem. Soc. 1964, 301. IUPAC. Nomenclature of Organic Compounds, 1979 Ed., Pergamon Press, p. 491. Preuss, F.R.; Orth, H. Pharmazie 1965,20, 698. Fuji, K.; Tanaka, K.; Li, B.; Shingu, T.; Sun, H.; Taga, T. Tetrahedron Lett. 1992,33, 7915. Appendino, G.; Barboni, L.; Gariboldi, P.; Bombardelli, E.; Gabetta, B. Viterbo, D. J. Chem. Soc., Chem. Commun. 1993, 1587. Ma, W.; Park, G.L.; Gomez, G.A.; Nider, M.H.; Adams, T.L.; Aynsley, J.S.; Sahai, O.P.; Smith, R.J.; Stahlhut, R.W.; Hylands, P.J.; Bitsch, F.; Shackleton, C. J. Nat. Prod. 1994, 57, 116.

49 41.

42.

43. 44. 45. 46. 47.

Appendino, G.; Cravotto, G.; Enrifi, R.; Gariboldi, P.; Barboni, L.; Torregiani, E.; Gabetta, B.; Zini, G.; Bombardelli, E. J. Nat. Prod. 1994, 57, 607. (a) Barboni, L.; Gariboldi, P.; Torregiani, E.; Appendino, G.; Gabetta, B.; Bombardelli, E. Phytochemistry 1994, 36, 987; (b) Chen, W.Z.; Zhou, J.Y.; Zhang, P.L.; Cheng, F.Q. Chin. Chem. Lett. 1993, 4, 699; (c) Hongjie, Z.; Takeda, Y.; Matsumoto, T.; Minami, Y.; Yoshida, K.; Wei, X.; Qing, M.; Handong, S. Heterocycles 1994, 38, 975. Ettouati, L.; Ahond, A.; Convert, O.; Laurent, D.; Poupat, C.; Potier, P. Bull. Soc. Chim. Fr. 1988, 749. Ettouati, L.; Ahond, A.; Convert, O.; Poupat, C.; Potier, P. Bull. Soc. Chim. Fr. 1989, 687. Balza, F.; Tachibana, S.; Barrios, H.; Towers, G.N.H. Phytochemistry 1991, 30, 1613. Chiang, H.C.; Woods, M.C.; Nakadaira, Y.; Nakanishi, K. J. Chem. Soc., Chem. Commun. 1967, 1201. Chen, S.-H.; Combs, C.M.; Hill, S.E.; Farina, V.; Doyle, T.W. Tetrahedron Lett. 1992, 7679.

48.

49. 50. 51. 52. 53. 54. 55. 56.

(a) Samaranayake, G.; Magri, N.F.; Jitrangsri, C.; Kingston, D.G.I.J. Org. Chem. 1991, 56, 5144. (b) Wahl, A.; Gu~ritte-Voegelein, F.; Gu~nard, D.; Le Goff, M.-T.; Potier, P. Tetrahedron 1992, 34, 6965. Ho, T.-J.; Lin, Y.-C.; Lee, G.-H.; Peng, S.-M.; Yeh, M.-K.; Chen, F.-C. Acta Cryst. 1987, C43, 1380. Zhang, H.; Takeda, Y.; Minami, Y.; Yoshida, K.; Matsumoto, T.; Xiang, W.; Mu, O.; Sun, H. Chem. Lett. 1994, 957. Chen, W.M.; Zhang, P.L.; Wu, B.; Zhang, Q.T. Chin. Chem. Lett. 1991, 2, 441. Harris, J.W.; Katki, A.; Anderson, L.W.; Chmurny, G.N.; Paukstelis, J.V.; Collins, J.M.J. Med. Chem. 1994,37, 706. Swindell, C.S.; Isaacs, T.F.; Kanes, K.J. Tetrahedron Lett. 1985,26, 289. Maier, W.F.; Schleyer, P.v.-R. J. Am. Chem. Soc. 1981,103, 1891. Appendino, G.; Lusso, P.; Gariboldi, P.; Bombardelli, E.; Gabetta, B. Phytochemistry 1992, 31, 4259. Appendino, G.; Ozen, H.~.; Gariboldi, P.; Gabetta, B.; Bombardelli, E. Fitoterapia 1993, 64, 47.

50 57. 58.

59. 60. 61. 62. 63. 64. 65. 66. 67. 68.

69. 70. 71. 72.

Appendino, G.; Ozen, H.~.; Gariboldi, P.; Torregiani, E.; Gabetta, B.; Nizzola, R.; Bombardelli, E. J. Chem. Soc. Perkin Trans. I, 1993, 1563. Barboni, L.; Gariboldi, P. Torregiani, E.; Appendino, G.; Cravotto, G.; Bombardelli, E.; Gabetta, B.; Viterbo, D. J. Chem. Soc. Perkin Trans. I 1994, 3233. Appendino, G.; Cravotto, G.; Enrifi, R.; Jakupovic, J.; Gariboldi, P.; Gabetta, B.; Bombardelli, E. Phytochemistry 1994,36, 407. Barboni, L.; Gariboldi, P.; Torregiani, E.; Appendino, G.; Gabetta, B.; Zini, G.; Bombardelli, E. Phytochemistry 1993, 33, 145. Zamir, L.O.; Nedea, M.E.; B61air, S.; Sauriol, F.; Mamer, O.; Jacqmain, E.; Jean, F.I.; Garneau, F.X. Tetrahedron Lett. 1992, 33, 5173. Appendino, G.; Gariboldi, P.; Pisetta, A.; Bombardelli, E.; Gabetta, B. Phytochemistry 1992, 31, 4253. Chu, A.; Davin, L.B.; Zajicek, J.; Lewis, N.G.; Croteau, R. Phytochemistry 1993, 34,473. Chmurny, G.N.; Paukstelis, J.V.; Alvarado, B.; McGuire, M.T.; Snader, K.M.; Muschik, G.M.; Hilton, B.D. Phytochemistry 1993,34,477. Appendino, G.; Varese, M.; Gariboldi, P.; Gabetta, B. Tetrahedron Lett. 1994, 35, 2217. Baxter, J.N.; Lythgoe, B.; Scales, B.; Scrowston, R.M.; Trippett, S. J. Chem. Soc. 1962, 2964. Cram, D.J.; Sahyun, M.R.V.; Knox, G.R.J. Am. Chem. Soc. 1962, 84, 1734. (a) Weinheimer, A.J.; Chang, C.W.J.; Matson, J.A. In Progress in the Chemistry of Organic Natural Products; Herz, W.; Grisebach, H.; Kirby, G.W., Eds.; Springer-Verlag, Vienna, 1979; vol. 36, p.285; (b) Wahlberg, I.; Eklund, A.-M. In Progress in the Chemistry of Organic Natural Products; Herz, W.; Kirby, G.W.; Moore, R.E.; Steglich, W.; Tamm, Ch., Eds.; Springer-Verlag, Vienna, 1992; vol. 59, p.141. Karlson, B.; Pilotti, A.-M.; S6derholm, A.-C.; Norin, T.; Sundin, S.; Sumimoto, M. Tetrahedron 1978, 34, 2349. Begley, M.J.; Jackson, C.B.; Pattenden, G. Tetrahedron Lett. 1985, 26, 3397. Zhang, Z.; Jia, Z. Phytochemistry 199~ 30, 2345. Burke, B.A.; Chan, W.R.; Honkan, V.A.; Blount, J.F.; Manchand, P.S. Tetrahedron 1980, 36, 3489.

51 73.

74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96.

Reviews: (a) Nicolaou, K.C.; Dai, W.-M.; Guy, R.K. Angew. Chem. Int. Ed. Engl. 1994, 33, 15. (b) Boa, A.N.; Jenkins, P.R.; Lawrence, N. Cont. Org. Synth. 1994, 1, 47. Li, B.; Tanaka, K.; Fuji, K.; Sun, H.; Taga, T. Chem. Pharm. Bull. 1993, 41, 1672. Errata: ibid. 1993, 41, 2200. Ettouati, L.; Ahond, A.; Poupat, C.; Potier, P. Tetrahedron 199~ 47, 9823. Magee, T.V.; Bornmann, W.G.; Isaacs, R.C.A.; Danishefsky, S . J . J . Org. Chem. 1992,57, 3274. Liang, J.; Kingston, D.G.I.J. Nat. Prod. 1993, 56, 594. Swindell, C.S.; Britcher, S.F.J. Org. Chem. 1986, 51,793. Gu~ritte-Voegelein, F.; Gu~nard, D.; Potier,P. J. Nat. Prod. 1987, 50, 9. Hegnauer, R. Phytochemistry 1988,27, 2423. Leete, E.; Bodem, G.B. Tetrahedron Lett. 1966, 3925. Platt, R.V.; Opie, C.T.; Haslam, E. Phytochemistry 1984,23, 2211. Zamir, L.O.; Nedea, M.E.; Garneau, F.X. Tetrahedron Lett. 1992, 33, 5235. Fleming, P.E.; Mocek, U.; Floss, H.G.J. Am. Chem. Soc. 1993,115, 805. Leete, E.; Kowanko, N.; Newmark, R. J. Am. Chem. Soc. 1975, 97, 6826. Fleming, P.E.; Knaggs, A.R.; He, X.-G.; Mocek, U.; Floss, H.G.J. Am. Chem. Soc. 1994,116, 4137. Fuji, K.; Tanaka, K.; Li, B.; Shingu, T.; Sun, H.; Taga, T. J. Nat. Prod. 1993, 56, 1520. Stierle, A.; Strobel, G.; Stierle, D. Science 1993, 260, 214. Am~bile-Cuevas, C.F.; Chicurel, M.E. Amer. Sci. 1993, 81,332. Py, S.; Khuong-Huu, F. Bull. Soc. Chim. Fr. 1993,130, 189. Kabayashi, T.; Kurono, M.; Sato, H.; Nakanishi, K. J. Am. Chem. Soc. 1972, 94, 2863. Takahashi, T. Yakugaku Zasshi 1931 51,401. Appendino, G.; Fenoglio, I.; Cravotto, G.; Varese, M.; Gariboldi, P.; Gabetta, B. Gazz. Chim. Ital. 1994, 124, 253. Magri, N.F.; Kingston, D.G.I.J. Org. Chem. 1986, 51,797. Appendino, G.; Gariboldi, P.; Gabetta, B.; Pace, R.; Bombardelli, E.; Viterbo, D. J. Chem. Soc. Perkin Trans. I 1992, 2925. (a) Kant, J.; Farina, V.; Fairchild, C.; Kadow, J.F.; Langley, D.R.; Long, B.H.; Rose, W.C.; Vyas, D.M. Bioorg. Med. Chem. Lett. 1994, 4, 1565; (b)

52

97.

98. 99. 100. 101. 102. 103. 104.

105. 106. 107. 108. 109.

110. 111. 112.

Ojima, I.; Fenoglio, I.; Park, Y.-H.; Pera, P.; Bernacki, R. Bioorg. Med. Chem. Lett. 1994, 4, 1571. Holton, R.A.; Kim, H.-B.; Somoza, C.; Liang, F.; Biedifer, R.J.; Boatman, P.D.; Shindo, M.; Smith, C.C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, K.K.; Gentile, L.N.; Liu, J . H . J . Am. Chem. Soc. 1994,116, 1599. Taylor, G.F.; Thornton, S.S.; Tallent, C.R.; Kepler, J.A.J. Label. Comp. Radiopharm. 1993,33, 501. Takahashi, T.; Ueda, K.; Oishi, R.; Minamoto, K. Chem. Pharm. Bull. 1958, 6, 728. Appendino, G.; Cravotto, G.; Gariboldi, P.; Gabetta, B.; Bombardelli, E.; Gazz. Chim. Ital. 1994, 124, 1. Taylor, D.R. West Aft. J. Biol. Appl. Chem. 1963, 7, 1. Appendino, G.; Tagliapietra, S.; Ozen, H.~.; Gariboldi, P.; Gabetta, B.; Bombardelli, E. J. Nat. Prod. 1993, 56, 514. Gunawardana, G.P.; Premachandran, U.; Buttes, N.S.; Whittern, D.H.; Henry, R.; Spanton, S.; McAlpine, J.B.J. Nat. Prod. 1992, 55, 1686. Hoke, S.H.; Cooks, R.G.; Chang, C.-J.; Kelly, R.C.; Qualls, S.J.; Alvarado, B.; McGuire, M.T.; Snader, K.M.J. Nat. Prod. 1994, 57, 277 and references therein. Grothaus, P.G.; Raybould, T.J.G.; Bignami, G.S.; Lazo, C.B.; Byrnes, J.B. J. Immunol. Meth. 1993, 5, 158. Appendino, G.; 0zen, H.~.; Fenoglio, I.; Gariboldi, P.; Gabetta, B.; Bombardelli, E. Phytochemistry 1993, 33, 1521. McLaughlin, J.L.; Miller, R.W.; Powell, R.G.; Smith Jr., C.R.J. Nat. Prod. 1981, 44, 312. Castellano, E.E.; Hodder, O.J.R. Acta Cryst. 1973,B29, 2566. (a) Georg, G.I.; Gollapudi, S.R.; Grunewald, G.L.; Gunn, C.W.; Himes, R.H.; Rao, B.K.; Liang, X.-Z.; Mirhom, Y.W.; Mitscher, L.A.; Vander Velde, D.G.; Ye, Q.-M. Bioorg. Med. Chem. Lett. 1993,3, 1345; (b) Hansen, R.C.; Holmes, R.G.; Shugert Jr., R.B.; Croom, E.M.; E1 Sohly, H.; Keener, H.M.; Cochran, K.D. presented at the Second National Cancer Institute Workshop on Taxol and Taxus, Washington, D.C.; September 1992. Gu, J.; Zheng, R.; Zhang, Z.; Jia, Z. Planta Med. 199~ 71,495. Appendino, G. Fitoterapia 1993, 64, 5. Bryan-Brown, T. Q.J. Pharm. Pharmacol. 1933, 5. 205.

53 113. (a) Tekol, Y. Planta Med. 1985,15, 357; (b) Tekol, Y.; Kameyama, M. Arzneim. Forsch. 1987,37, 428. 114. Baureis, R.; Steiert, W. Arzneim. Forsch. 1959, 9, 77. 115. Rowinsky, E.K.; McGuire, W.P.; Guarnieri, T.; Fisherman, J.S.; Christian, M.C.; Donehower, R.C.J. Clin. Oncol. 1991,9, 1704. 116. Chaudhury, R.R.; Sakesena, S.K.; Garg, S.K.J. Reprod. Fertil. 1982, 22, 1512. 117. Khanna, U.; Garg, S.K.; Vohora, S.B.; Walia, H.B.; Chaudhury, R.R. Ind. J. Med. Res. 1969,57, 237. 118. Vohora, S.B.; Kumar, I. Planta Med. 1971 2,100. 119. Sceopul, T. Fr. Pat.l,525,945 (1968). Chem. Abstr. 1969, 71, 42169h. 120. Nielsen, M.; Frokjaer, S.; Br~iestrup, C. Biochem. Pharmacol. 1988, 37, 3285 121. Parveen, N.; Taufeeq, H.M.; Khan, N.U.-D. J. Nat. Prod. 1985, 48, 994. 122. Parmar, V.S.; Vardhan, A.; Taneja, P.; Sinha, R.; Patnaik, G.K.; Tripathi, S.C.; Boll, P.M.; Larsen, S. J. Chem. Soc. Perkin Trans. I 1991 2687. 123. Burke, M.J.; Siegel, D.; Davidow, B. N.Y. St. J. Med. 1979, 1576. 124. Hausen, B.M. Holzarten mit Gesundheitsschddigenden Inhaltsstoffen; Stuttgard, 1973. 125. Yu, X.; Li, P.; Dong, X.; Dong, L.; Shu, S. Hangzhou daxue Xuebao, Ziran Kexueban 1986,13,347; Chem. Abstr. 106, 33359a. 126. Tyler Jr., V.E.J. Am. Pharm. Ass. 1960, 49, 683. 127. Hegnauer, R. Pharm. Weekblad 1959, 94, 241.

The Chemistry and Pharmacology of Taxol and its Derivatives V. Farina, editor 9 1995 Elsevier Science B.V. All rights reserved

55

2 THE STRUCTURAL ELUCIDATION OF T A X O I D S Giovanni Appendino Dipartimento di Scienza e Tecnologia del Farmaco, Via di Giuria 9, 10125 Torino, Italy

2.1. I N T R O D U C T I O N

The taxane skeleton can r e a r r a n g e under a variety of experimental conditions (acids, bases, light), and the early work based on degradative chemistry gave no indication on its actual constitution. Puzzling [YV features together with the difficulties in assessing purity concurred to delay the structure elucidation of taxoids until the advent of NMR spectroscopy. The application of powerful two-dimensional techniques (COSY, NOESY, HMBC etc.) and the availability of a wealth of published information have made the structure elucidation of new taxoids almost trivial, provided the compound is available in sufficient quantity and purity for the application of these techniques. 2.2. M A S S S P E C T R O S C O P Y

Mass spectroscopy (MS) of taxoids has been used to obtain information on the molecular weight and to identify the acyl groups bound to the diterpenoid

55 core. Soft ionization techniques (FAB, DCI, TSP) have been used [1]. Thus, the chemical ionization spectra (NH3) of taxols shows a parent ion (M + NH4)+, and a prominent peak at m/z 586 [M + NH4 - side chainH] +, resulting from the loss of the aminoacid side chain [2]. The constitution of the latter can be elucidated by the analysis of the fragmentation pattern [3], but MS had so far played only a marginal role in the analysis of the diterpenoid core of taxoids. The most prominent peaks in the spectrum of the semisynthetic taxane tetraacetyltaxinol (Figure 1) have been tentatively identified [4], but study of closely related taxanes failed to confirm the generalization of the proposed fragmentation pattern.

0

AcO

OAc

H

-

_

"OAt_,

OAc 2.1.1 Tetraacetyltaxinol Figure 1

2.3. UV AND CD (ORD) S P E C T R O S C O P Y

The most important chromophore in the diterpenoid moiety of taxoids is the enone system found in compounds of the 10- and 13-oxo-A 11 type. In taxanes, these systems can be distinguished on the basis of UV data (Figure 2). The 13oxo-A 11 system shows an anomalous n->~* absorption band at 275-285 nm (a ca. 5,000-6,000) [5], rather different from the value calculated from Woodward's rules (Xmax ca. 255 nm). Comparison with related compounds highlights the role of ring strain and of the g e m - d i m e t h y l group at C-15 bathochromic shift (ca. 7 nm) by the C-1 hydroxyl groups was The 10-oxo-A 11 system, on the other hand, does not properties, since its maximum (Xmax 240-250 nm, a c a .

for this effect. A also observed [5]. show unusual UV 3,000) is in good

agreement with the value calculated from Woodward's rules [6]. In compounds of this type, the chair-boat conformation of the cyclooctane B ring allows little overlap between the bridgehead double bond and the C-10 carbonyl, since the

57 corresponding u orbitals are almost orthogonal to each other [7]. Thus, conjugation is attained only if the conformation of ring B is significantly changed in the fragment C-8/C-11. Taxanes and 2(3->20)abeotaxanes of the 13-hydroxy (acyloxy)- A l l type show an anomalous n->u* absorption (~ ca. 10,000) at 210-230 nm (expected value ca. 190-200) [5], showing t h a t the bridgehead double bond has an anomalous electronic distribution, possibly r e l a t e d to the gem-dimethyl substitution at C-15 and to its rigid trans-cyclodecene (dodecene) nature. This anomalous absorption is responsible for an intense and positive Cotton effect, amenable to octant analysis [8]. The results of this configurational assignment are supported by comparison with the CD spectra of chiral olefins (transcyclodecene and trans-cyclooctene) and with the results obtained on taxoids with other techniques (NMR, X-rays, Horeau methods). The A4,2~ is responsible for a weak negative Cotton effect, which is of no practical use, being overshadowed by the strong positive Cotton effect ot the All system [8]. The C-9 keto group of baccatin III gives rise to a Cotton effect at 304 nm, whose observed negative sign is predicted by the carbonyl octant rule [8]. The benzoate sector rule and the dibenzoate chirality rules were applied to taxanes of the A4(20)-5a benzoyloxy type and 0(9), O(10)-dibenzoyl type, respectively [9, 10], and these results were used for the structure elucidation of taxinine [9].

HO

OH

H

OH 3.1.1 4(20)-Dihydro-5-deoxytaxicin I )~m~x = 283 nm (e = 5,700) [5]

"

H

3.1.2 Taxuyunnanine D ~ m a x -- 248 nm (e = 2,275) [6b]

Figure 2: UV differences between 13-oxo and ll-oxo All-enones The absolute configuration at C-1 of taxoids is opposite to the one found in their alleged biogenetic precursors (verticilloids) isolated so far from plants (see section 1.6.1). However, all evidence from CD studies (olefin and carbonyl octant

58 analysis, benzoate sector and dibenzoate chirality rules) fully confirms such absolute stereochemistry. 2.4. IR S P E C T R O S C O P Y

The IR spectra of taxoids do not show any unusual absorption band. Owing to extensive inter- and intramolecular hydrogen bonding, the hydroxyl and carbonyl regions generally show broad bands, especially in the solid state. 2.5. N M R S P E C T R O S C O P Y

Despite the importance of NMR spectroscopy for the structure elucidation of taxoids and the impressive technical advances made in the last decade, no recent review on the NMR features of taxoids is available. In 1963, Nakanishi discussed in detail the 1H-NMR spectrum of taxinine and some of its derivatives [11]. Although based on a wrong stereostructure (C3~H), most of these assignments are correct. In 1967, Lythgoe made extensive use of 1H-NMR data to correctly assign the stereochemistry of taxicin I and II [12], confirming most of the assignments and observations made by Nakanishi [11]. In 1980, Miller reviewed the available 1H-NMR data on taxoids [13], and in 1983 Rojas discussed the general features of the 13C-NMR spectra of several structural types of taxanes [14, 15]. In the last few years, many papers have discussed in detail the NMR spectra of Taxol| and related compounds [16-23]. All resonances were assigned, and solvent-related conformational changes at the side chain were noted [20-23]. Until the advent of two-dimensional techniques, most assignments in the 13C-NMR spectra were carried out by analogy or left uncertain. Reference will be made here mainly to data derived from fully assigned spectra. In order to better appreciate the stereochemical information derived from the NMR spectra, a section on the general conformational features of taxoids is presented next, before the full discussion of the NMR data. 2.5.1. Conformational Aspects of the Taxoidic Skeleta The shape of the diterpenoid core of taxoids is dictated mainly by the conformation of ring B. The latter is a cyclooctane in taxanes, a cycloheptane in

ll(15->l)abeotaxane, a cyclodecane in 2(3->20)abeotaxanes, and a bicyclo[3.3.0]

59 octane in 3,11-cyclotaxanes (see Figure 7 in chapter 1). More than one conformation is possible for ring B, except in 3,11-cyclotaxanes, where the presence of a t r a n s a n n u l a r bond prevents conformational mobility. Several conformations are possible for cycloheptane, cyclooctane and cyclodecane, but the junction with ring A and ring C makes only a few of them possible for ring B of taxoids. The major variation occurs along the C-9/C-10 bond, the only one between carbons not shared with ring A or ring C. Among the staggered conformations around C-9/C-10, two conformers are possible, and they are characterized by a syn or an anti relationship between the oxygen functions at C9 and C-10 (Figure 3, A and B). An eclipsed conformation with the oxygen syn (C) is also possible. The staggered, oxygen syn conformation around C-9/C-10 (A) is typical of taxanes, with the notable exception of taxagifine and its derivatives, which adopt the eclipsed conformation C [24]. Both staggered conformations A and B have been found in ll(15->l)abeotaxanes, whereas 2(3->20)abeotaxanes adopt the staggered/oxygen-syn conformation (A) typical of taxanes. All ananchomeric (i.e. conformationally locked) taxoids with the C-9/C-10 staggered, oxygen-anti conformation (B) have an extra oxygen bridge, between C-10 and C15 or C-13 and C-15 (Figure 4) [25, 26].

H9

'R

OR

R

H9Ho

Hlo

~(c-9/c4o)=6oo A

OR

Hlo ~(c-9/c-1 o)=-oo o B

~(c-9/c-~o)=12o o C

Figure 3: Newman projections along the C-9/C-10 bond in the three major conformations of taxoids.

No universally accepted denomination for the topological forms of medium-sized rings exists, and pictorial stereoviews of taxoids are difficult to draw and to interpret. The arrangements A and B correspond to a chair-boat and a chair-chair conformation for ring B in taxoids, and to a twist-boat and a twistchair conformation for ring B in ll(15->l)abeotaxanes. The Bucourt notation [27] makes reference to the succession of the endocyclic torsion angles, and seems more meaningful in this context [26].

60 Ring C is relatively rigid in oxetane-type taxoids, and adopts a sofa conformation with C-7 as the flap (Figure 5, A). The c h a i r and the boat c o n f o r m a t i o n are i n s t e a d

possible in A4(20)-taxoids, d e p e n d i n g on the

conformation of ring B or the presence of an additional C-3/C-11 bond (Figure 5, B and C) [28, 29]. Ring A adopts a distorted boat conformation in taxanes and

2(3->20)abeotaxanes, and an envelope conformation in ll(15->l)abeotaxanes.

~Ac

10 ~ - - "/'~O

AcO

_ ~ 0 OBz OAc

OAc

0

0 OBz

5.1.1

5.1.2

Figure 4: Ananchomeric taxoids with a staggered, oxygen-anticonformation around the C-9/C-10 bond. 2.5.2. Proton and Carbon NMR Assignments of the Diterpenoid Core For the sake of clarity and practicality, the NMR features of the proton(s) and carbon referring to the same position of the taxoidic skeleton will be discussed under the same heading. Throughout this section, bold numbers refer to compounds whose spectroscopic data are presented in section 2.5.4.

H H

O 02 Me

Ac

02 H713

H5

HFo~ A

02

Me H71~ ~

OR

HFa

//

B

Me H7~

~m

H7c~

C

Figure 5: Conformation of ring C of taxoids C-l:

D e p e n d i n g on the s t r u c t u r a l type of taxoids, C-1 can be a

h y d r o x y l a t e d q u a t e r n a r y carbon, an aliphatic m e t h i n e , or an aliphatic quaternary carbon, resulting in diagnostic resonances at 5 75-80, 40-50 or 60-70,

61 respectively. C-1 is an aliphatic quaternary carbon in 11(15->1)abeotaxanes (13 and 14), and its downfield resonance (5 60-70) is diagnostic for this type of taxoids [29b]. This chemical shift, unusual for a non-oxygenated carbon, might be the result of linear strain, since in these compounds some of the carboncarbon bonds centered at C-1 are significantly longer than normal [29b]. Owing to slow conformational equilibration, the signal of C-1 in ll(15->l)abeotaxanes is sometimes very broad or even undetectable at room temperature [26]. The C-1 hydroxyl is not acylated u n d e r s t a n d a r d or even forcing conditions, but acylation can be attained in situ (i.e. in the NMR probe) with the powerful acylating reagent trichloroacetyl isocyanate (TAI) [29b]. Several hours are required for the reaction, which causes a dramatic downfield shift on H-1413 (>1 ppm), and smaller shifts on the gem-dimethyls at C-15, H-14a and H-2 (Figure 6). This p a t t e r n of A5 allows a straightforward distinction between C-1 hydroxylated taxanes and C-15 hydroxylated 11(15->1)abeotaxanes. These two classes of compounds show similar 1H NMR spectra, and the rigorous establishment of the carbon connectivity via long-range 1H-13C-correlations can be difficult because of the fluxional behavior of 11(15->1)abeotaxanes [29]. However, acylation of the tertiary hydroxyl of these compounds has only a small effect on the resonance of H-14[~, but causes a large paramagnetic shift on the geminal methyls, a diamagnetic shift on H-913, and has a negligible effect on H-2 (Figure 6).

AcO

OAc

~ AcO .... +1.44

H

+0.29 H

L ~

-

:

c

L

~

OH ,~'~ n OAc BzO H +0.38

5.2.1 Baccatin VI

BzO

H -0.20 OAc " OAc

AcO,,. 0

H ~ ~ ' ~ ~ ~ / _ H~ OAc OAc +o.46H0~ +0.58

O

+0.14

5.2.2 Isoabeobaccatin VI

Figure 6: A (TAI) observed upon acylation of the tertiary hydroxyls of baccatin VI and isoabeobaccatin VI [29b].

62 H-1 is almost orthogonal to H-14a, and J l,14a is ca. 0 Hz. Coupling is instead observed with H-14[~ (ca. 7 Hz) and H-2~ (ca. 2 Hz). C-2: In C-2 oxygenated taxoids, the assignment of H-2 is straightforward, owing to a peculiar multiplicity pattern (J1,2 c a . 2 Hz; J2,3 ca. 7 Hz), easily distinguished from that of the other oxymethines. In taxoids of the 4,20-epoxide type, J2,3 is smaller (3-4 Hz) (1), but the splitting pattern of H-2 is still diagnostic, as is in taxagifine-type taxoids, where J2,3 is c a . 10 Hz (15). In taxanes having a C-2, C-9, C-10, C-13 hydroxylation (acyloxylation) pattern, H-2 is generally the most shielded of these oxymethines, regardless of the acyl residue at C-2 (5.2.1, Figure 7). In the corresponding l l ( 1 5 - > l ) a b e o t a x a n e s , H-2 is instead the most deshielded of these signals (5.2.3, Figure 7) [26, 30]. This observation has diagnostic value, although exceptions exist in taxagifine-type taxoids (15).

AcO

OAc

AcO ~,

OAc ~ OAc

6.31/ \lib ,/~.~ 6.13'~,,~ AcO'"

4

HO

% ,,

O

AcO....~

=.

6Bz 5.2.1 Baccatin VI [26]

Hd \

O ~6.36 OAc OBz

5.2.3 Abeobaccatin VI [30]

Figure 7: Ring A/B oxymethine resonances in baccatin VI and abeobaccatin VI

In C-2, C-10 di-oxygenated taxoids, the a s s i g n m e n t of C-2 is not straightforward, since its chemical shift (ca. 70-75 ppm, depending on its acylation state and that of C-l) is similar to that of C-10. However, since the signals of H-2 and H-10 are very well separated, two-dimensional techniques or selective decouplings allow an unambiguous assignment. When C-2 is a methylene, the C-1 hydroxyl has a strong deshielding effect (ca. 10 ppm, cf 9 vs. 11 ) on its resonance. Only a moderate effect ( ca. 2 ppm, cf 6 vs. 7) is instead observed when C-2 is an oxymethine, presumably because of a shielding 7-gauche interaction between the vicinal oxygens.

63 C-3: In 2(3->20)abeotaxanes, C-3 is a methylene, and the appearance of an isolated AB system with lines at 8 2.60-2.80 and 1.60-1.80 is a diagnostic

feature of taxine A derivatives (see 17). The corresponding 13C resonance is found at 8 ca. 35, close to the C-6 triplet. In taxanes and 11(15->1)abeotaxanes, C-3 is an aliphatic methine and H-3 is a doublet, resonating at relatively low field in compounds of the A4(20)-type (8 c a . 3.5) and 9-oxo-4a-acyloxy-5,20oxetane type (baccatin III/V derivatives) (8 ca. 3.5-4.0). In oxetane-type taxoids lacking a 9-oxo group (baccatin IV,VI,VII derivatives) or a 4-acetyl (see 4), H-3 resonates at 8 2.5-3.2, and the same chemical shift range is observed in taxoids of the 4(20)-epoxide type (see 1). The resonance of C-3 is found at 8 35-45 in compounds of the A4(20)-type, and at 8 45-50 in compounds of the oxetane type. An unusual value for C-3 (8 ca. 59 !) was found in A4(20)-taxoids bearing an amethylbutyrate ester at C-2 [15]. This remarkable effect was rationalized in terms of a conformational change in ring B that moves C-3 away from the 11-12 double bond [15]. However, the oxygenation pattern of these compounds should be confirmed by modern NMR techniques, since several other resonances are quite unusual for the proposed structure. In 3,11-cyclotaxanes (16), C-3 is a quaternary carbon, resonating at relatively low field (8 ca. 60) for a nonoxygenated tetrahedral carbon. As with C-1 in 11(15->1)abeotaxanes, linear strain may be responsible for this unusual chemical shii~ value. C-4: In all but one of the naturally occurring taxoids, C-4 bears no hydrogens. In compounds of the A4(20)-type, C-4 resonates at 8 140-150,

depending on the oxygenation state of C-2 and C-5. The corresponding resonance in compounds of the oxetane- and epoxide-type are 8 ca. 80 and 60 respectively. Hydrolysis of the 4-acetoxy group causes an expected upfield shift on C-4 (ca. 6 ppm, cf 4 and 5). C-5: In all natural taxoids C-5 is oxygenated, and the splitting pattern of

H-5 can give important information on the stereochemistry at this carbon as well as on the conformation of ring C. In taxoids of the A4(20)-type, H-5 is always ~, and its splitting pattern depends on the conformation of ring C. In A4(20)_ taxenes, ring C has a chair conformation (B, Figure 5), and H-5 is equatorial (J5,6a=J5,6~1)abeotaxanes both the chair (B) and the boat conformation (C) have been detected [28, 29]. In the latter, Js,6a=J5,6~=ca. 5 Hz. In A4(20)-3,11-cyclotaxenes (see 16), ring C has a twist-boat conformation, and J5,6a=J5,6~=ca. 9Hz [31]. In taxoids of the 4,20 epoxide type, H-5 resonates at unusually high fields, yielding misleading information on the acylation state of

54 the C-5 hydroxyl (see 1). In oxetane-type taxoids, H-5 is a and ring C has a sofa conformation (A, Figure 5). In these compounds, H-5 resonates as a doublet of doublets (J5,6a= ca. 9 Hz, J5,6~=ca. 2 Hz). Opening of the oxetane via anchimeric assistance from the 4-acetyl inverts the stereochemistry at C-5, and H-5 turns into a narrow triplet, with J5,6a and J5,6~80. In 9,10-dihydroxylated taxanes, H-9 resonates at higher field than H-10 (8 ca. 4.3 and 5.0, respectively), and a similar trend is observed in 11(15>l)abeotaxanes

(14). This observation is useful in establishing the acylation

pattern of O(9),O(10)-monoesters [36]. In 9,10-dihydroxylated taxoids, the vicinal hydroxyls are always t r a n s (~ and ~, respectively), but the value of J9,1o depends on the conformation of ring B, which dictates the sign of the torsion angle C-8/C9/C-10/C-11. When this fragment is staggered and the angle is positive, H-9 and H-10 are t r a n s - d i a x i a l , and J9,1o is around 10 Hz (Figure 3, A). Rotation around the C-9, C-10 bond and the attainment of a negative value for the endocyclic torsion angle around these carbons makes H-9 and H-10 t r a n s - d i e q u a t o r i a l , and therefore J9,10 decreases to ca. 4 Hz (Figure 3, B), as observed in some 11(15->1) abeotaxanes

[28, 29a]. Small values of J9,10 (ca. 3 Hz) are also typical of

taxagifine-type compounds (oxygen bridge between C-17 and C-12, see 15), where the fragment C-8/C-9/C-10/C-11 is in an eclipsed conformation (~ ca. 120~ and H-9 and H-10 are anticlinal (Figure 3, C). C-10: In all natural taxoids C-10 is an oxymethine, whose chemical shift (5 65-75) is little affected by acylation or by the presence of a keto group at C-9. A change in hybridization of C-11, as in 3,11-cyclotaxanes (see 16), shifts C-10 downfield (8 80-82). Epimerization at C-7 of baccatin III to baccatin V causes downfield shifts at H-10 (ca. 0.5 ppm) and C-10 (ca. 3 ppm) (cf. 18 and 19) and a similar, but opposite, effect on C-12. The long-range effects are difficult to rationalize, but probably reflect subtle conformational differences and/or hydrogen bonding patterns. An even more marked downfield shift (AS ca. 5 ppm) on the chemical shift of C-10 was observed between 10-oxo-derivatives of the baccatin III and baccatin V series, suggesting better conjugation between the C10 carbonyl and the 11-12 double bond in baccatin V derivatives [37]. C - 1 1 : C - 1 1 is a tertiary alcohol in taxagifine derivatives (8 c a . 80), a quaternary aliphatic carbon in 3,11-cyclotaxanes (8 ca. 55) and an olefinic carbon in all the other taxoids, resonating at 8 130-135 (150-155 when a 13-oxo group is present). In 1 3 - h y d r o x y - 1 1 ( 1 5 - > 1 ) a b e o t a x a n e s , C-11 is slightly more deshielded than in the corresponding taxanes (AS ca. 2 ppm, cf. 5 and 14). The chemical shift

55 of C-11 is affected by esterification of the allylic hydroxyls at C-10 and C-13, which causes an upfield shift (ca. 3 and 1 ppm, respectively). C-12: C-12 is an aliphatic methine (5 ca. 50) in 3,11-cyclotaxanes, a quaternary oxygenated carbon in taxagifine derivatives (5 ca. 90) and an olefinic carbon (5 135-140) in all the other structural types oftaxoids. C-13: Like C-9, C-13 can be an oxymethine, a carbonyl, or an aliphatic methylene. In 13-hydroxy (acyloxy) taxoids, the chemical shift of C-13 depends on the structural type, and in l l ( 1 5 - > l ) a b e o t a x a n e s the resonance for this carbon is found at lower field than in taxanes (5 77-80 vs. 67-72, cf. 14 and 5). The carbonyl of All-13-oxotaxanes resonates around 199 ppm, as expected for a 2-cyclohexenone carbonyl, but the chemical shift of the carbonyl of 13-oxo-3,11cyclotaxanes (5 214-216, see 16) is rather downfield for a cyclohexanone carbonyl. In natural taxoids, H-13 is almost always ~, and its signal is rather broad, due to allylic coupling with H-18 (J ca.1 Hz). In taxanes, J13,14~ is ca. 9 Hz, whereas the value of J13,14a varies considerably (3-9 Hz), depending on the conformation of ring A. In l l ( 1 5 - > l ) a b e o t a x a n e s , J13,14~ is generally smaller (ca. 7 Hz) than in the corresponding taxanes [26]. Only one example of natural taxoid with a 13~oxygen function has been reported to date. In this compound, values of ca. 0 and 1.8 Hz were observed for J13,14a and J13,14~, respectively [26]. C-14: C-14 can be an aliphatic methylene (5 35-45, depending on the functionalization of its a-carbons)or an oxymethine (5 ca. 70). The geminal H-14 protons are generally well separated, and H-14a, pointing toward the concave face of the molecule, is the most shielded. A C-13 oxo group deshields H-14a more t h a n H-14~, and consequently, in some taxicin I derivatives, the resonances of H-14a and H-!4~ are close to each other. H-14a is also deshielded by an oxygen function at C-1 and, in oxetane-type taxoids, H-14a,~ are sometimes almost isochronous, or their chemical shift values can sometimes be reversed (see 4). C-15:C-15 is an aliphatic quaternary carbon (5 37-43, depending on the presence or absence of a hydroxyl group at C-l) in all taxoids, except in 11(15> l ) a b e o t a x a n e s , where this carbon is oxygenated (5 75-79). In these compounds, a strong intramolecular hydrogen bond between the hydroxyl at C-15 and the oxygen function at C-10 exists, and the C-15 hydroxyl proton resonates as a singlet, unaffected by dilution, at 5 2.40-2.50 [29b]. In taxagifin-type taxoids, C15 is aliphatic, but the oxygen functions on the a-carbons (C-11 and C-17) shift its resonance downfield to ca. 50 ppm (see 15).

67

C-16, C-17: In taxanes, the proton and carbon signals of the g e m dimethyls are often well differentiated. The C-17 methyl, facing ring A, is generally the most shielded in the 1H NMR spectrum, but an opposite relationship is observed in the 13C NMR spectrum. A5 of up to 0.60 ppm (1H NMR) and 15 ppm (13C NMR) have been reported (see 6 and 7). A keto group at C-9 has a shielding effect on H-16, but has an opposite effect on C-16. This upfield shiit of H-16 is responsible for the small A5 between the geminal methyls in the 1H NMR spectra of baccatin III/V derivatives. Furthermore, the phenylisoserine residue at C-13 has a deshielding effect on H-17, and consequently in taxols H-17 resonates more downfield than H-16, in contrast with what observed in the other types of taxoids (see 18 and 19). The C-1 hydroxyl shifts H-16 upfield and H-17 downfield, and both C-16 and C-17 upfield, as a result of a 7-gauche shielding interaction (see 6 vs. 7 and 9 vs. 11). X-ray analysis has shown that in taxanes the OH/C-I/C-2 bond angle is smaller than the tetrahedral value (100-105 ~ [14]. As a result, the hydroxyl bisecting the C-16/C-15/C-17 angle is closer to C-16 than C-17, and the T-gauche effect is larger for C-16 than C-17. This might also explain why, upon acylation of the C-1 hydroxyl, H-16 suffers a larger downfield shift than H-17 (see Figure 6). The singlets for H-16 and H-17 are generally broader than the H-19 singlet. To explain this observation, Nakanishi suggested the presence of a weak coupling between the geminal methyls [11], but two-dimensional techniques (COSY) show little, if any, coupling between these hydrogens. The singlet for H16 is often taller than the one for H-17, especially when a C-1 hydroxyl is present. It is likely than both gem-dimethyls of taxanes are subject to long-range couplings but, apart from H-l, the protons involved have not yet been identified. C-18: In the 1H NMR spectra of taxoids of the All type, the 18-methyl is the one generally resonating at the lowest field. The presence of a keto group at C-10 or C-13 does not cause an appreciable downfield shift, but acylation of the C-10 hydroxyl causes a downfield shift (0.10-0.15 ppm) that has diagnostic value for the location of the acylation site in 0(9), O(10)-monoesters [36]. In oxetanetype taxoids, the presence of the aminoacid side chain at C-13 causes a marked upfield shift on H-18 (5 = 2.01 in baccatin III, but 1.79 in Taxol| which resonates close to H-19 (see 18 and 19). In compounds of this type, a change in solvent polarity has little effect on the chemical shirt of H-18. This is surprising, since changes are observed within the hydrogens of the side chain, which is spatially close (NOE effect) to H-18.

58 Allylic coupling is observed between the 13~ hydrogen and the 18-methyl (4J13,18 c a . 1.0 Hz). In 3,11-cyclotaxanes (see 16), the 18-methyl resonates as a doublet (J ca. 7 Hz) and this is a diagnostic feature for this class of taxoids, since l l , 1 2 - d i h y d r o t a x a n e s are u n k n o w n as n a t u r a l products. In A ll-taxenes, the resonance of C-18 has a fairly narrow chemical shift range (12-15 ppm). The oxidation state of C-13 has little effect, b u t contraction of ring A from a cyclohexene to a cyclopentene, as in l l ( 1 5 - > l ) a b e o t a x a n e s , shifts the allylic methyl upfield (AS ca. 3 ppm), as shown by comparison of the 13C NMR spectra of 5 and 14. A similar, but not so large, effect is also observed in the 1H NMR spectra (5 =2.01 for H-18 in baccatin VI, 5.2.1, but 1.73 in abeobaccatin VI, 5.2.3, Figure 7)[30]. C-19. The chemical shift of the 19-methyl is affected by oxygen functions at C-7, C-9 and C-20, and its 1H and 13C resonances vary over a wider range than the ones for the other methyl groups. An a or ~ C-7 hydroxyl has little effect on H-19, but a 7~-hydroxyl causes a large upfield T - g a u c h e shii~ (up to 7 ppm) on C-19 (see 18 and 19). This allows a clearcut distinction between baccatin III and baccatin V derivatives, since C-19 resonates at ca. 10 ppm when the 7-OH is ~, and at c a . 15 ppm when the 7-OH is (~ (cf 18 and 19 ). Comparison of the resonance of C-19 for baccatin III (5 9.5) and baccatin VI (5 12.7) shows that, "when compared to a C-9 acetoxyl, the C-9 keto group has a shielding effect on C19. The presence of a 5,20-oxetane ring has little effect on the chemical shii~ of C-19, but causes a marked downfield shift (ca. 0.50 ppm) on H-19. Since also a 9-oxo group has a deshielding effect on H-19, in baccatin III derivatives H-19 is the most deshielded of the three non-allylic methyls (5 ca. 1.60, see 2, 3, 18 and 19). On the contrary, in A4(20)-taxoids, H-19 is generally the most shielded methyl, and resonates at fields as high as 0.80-1 ppm (see 6-12). A 4,20~-epoxide has also a deshielding effect on H-19, but not so large as a 5,20-oxetane ring (cf. 1 and 10). In 19-oxygenated taxoids, 2j 19a,~ is ca. 12 Hz (see 15). The AB system of H19a,~ is easily distinguished from the AB system of the oxymethylene protons at C-17 (taxagifine derivatives) or at C-19 (oxetane-type taxoids). Indeed, the presence of a cyclic structure in these latter compounds decreases the geminal coupling to ca. 8 Hz. C-20:

C-20 is an olefinic m e t h i n e in 2 ( 3 - > 2 0 ) a b e o t a x a n e s ,

and a

functionalized methylene, resonating as an AB system, in all the other structural types of taxoids. The separation of the AB system can yield information on the

59 stereochemistry at C-4 (4,20-epoxides), at C-7 (5,20-oxetanes) or on the functionalization of C-2 and C-5 (A4,20-taxoids). Thus, in the n a t u r a l 4~,20 epoxides, H-20a,~ are well separated (AS > 1 ppm), due to the downfield shift of the hydrogen facing ring B (5 ca. 3.50). In the isomeric and u n n a t u r a l 4a,20 epoxides, H-20cz,~ are instead much closer together (AS 666 mg/ml at ambient temperature. The half life of 3.3.10 was determined to be approximately 4.0 h and 1.1 h in pH 7.4 buffer and rat plasma, respectively. The authors have not reported any in vivo antitumor activity, but the approach seems very promising. Paclitaxel C-7 Esters: In general, C-7 esters have found little utility as watersoluble prodrugs of paclitaxel. To date, several C-7 derivatives have been reported [30] and in most cases they have been the counterparts of their corresponding C-2' derivatives. Their poor prodrug properties have been attributed to enhanced in vivo stability cleavage.

toward esterases and hydrolytic

For example, the cationic water-soluble derivative 3.1.11(Figure 3) had a half life of 378 h at pH 3.8 compared with a half life of of 96 h for its corresponding C-2' ester derivative. A similar trend was observed at physiological pH (7.4), where 3.1.11 had a half life of 34 h compared to 6 h for its C-2' counterpart. Half lives in human plasma for 3.1.11 and its C-2' counterpart were reported to be 3 h and C-1 benzoyl shift under basic conditions, leading, after t r e a t m e n t with carbon disulfide and methyl iodide, to 1-benzoyl-2-xanthyl derivatives. A re-examination of the earlier work [16b] verified that this is indeed the case in the baccatin series also (Scheme 1). Thus, 2.1.3 represents the first example of a C-1 acylated baccatin

168 III derivative. C-2 deoxygenation led to the deoxy analog 2.1.4. Similar results have been reported by the Kingston group as well [16c]. C-1 substitution does not imperil C-13 side chain introduction for biological evaluation. As outlined in Scheme 2, xanthate 2.1.3 was desilylated and then selectively resilylated at C-7 to give 2.1.7./~cylation according to the method of Holton [18], employing ~-lactam 2.1.10 as the side chain source, gave the desired C-1 functionalized paclitaxel derivative 2.1.9, after a final desilylation [19]. In search of a suitable C-1 protecting group, Chen and co-workers were led to utilize the novel dimethylsilane (DMS) group. Apparently, introduction of the bulkier trimethylsilyl group was difficult. As illustrated in Scheme 1, DMS was successfully introduced to the C-1 position of baccatin derivative 2.1.2 to give 2.1.5 in almost quantitative yield. Selective removal of DMS from C1 was accomplished with tetrabutylammonium fluoride at 0 ~ [20].

.o ....,,co 2o. TESO. . . . . . . . . . HO BzO

HO ~c

2.1.1 R=H -~ i 2.1.2 R=TES J

MeS 2.1.3

l iii AcO TESO....

O OTES

AcO

~0

TESO. . . . . . . . . .

20TES

Me2HSiO BzO 2.1.5

2.1.4

Conditions: (i) 5 equiv TESC1, imidazole, DMF, rt, 87%; (ii) Nail, THF+CS2, 70 ~ then MeI, 56%; (iii) 2 equiv Bu3SnH, AIBN, PhMe, 100 ~ 92%; (iv) 3 equiv Me2HSiC1, imidazole, DMF, 0 ~ 97%; (v) Bu4NF, THF, 0 ~ 84% of 2.1.2. [TES=triethylsilyl]

169 The significance of DMS protection of the C-1 hydroxyl group in the selective deacetylation at C-4 will be discussed in section 5.2.2. C-1-Benzoyl-2-deoxybaccatin 2.1.4 was converted to the corresponding C7 silylated analog 2.1.12 via a desilylation/mono-silylation sequence. The paclitaxel side chain was then attached onto 2.1.12 using Holton's protocol, to give 1-benzoyl-2-debenzoyloxytaxol, 2.1.14, after standard deprotection (Scheme 3) [19]. [ S c h e m e 21

AcO ~., RO ....\ \ BzO

Bz,

O ,~ OR'

P h ~ O

iii

=

AcO

MeS2CO 2.1.3 R=R'=TES ~ i 2.1.6 R=R'=H 2.1.7 R=H, R'=TES ~ i i

....

OR

O TESO,",,

-

O OR

NH O

Ph ],-" N. oJBz

O

BzO =MeS2CO

OAc

"

2.1.8 R=TES ) i 2.1.9 R=H

2.1.10

Conditions: (i) Py, 48%HF, CH3CN, 5 ~ 2.1.3 to 2.1.6, 76%; 2.1.8 to 2.1.9, 84%; (ii) TESC1, imidazole, DMF, 0 ~ 86%; (iii) LiHMDS, THF, -45 ~ then 2.1.10, 86%. [ Scheme 3 /

AcO RO ....

O OR'

Bz" NH

AcO ~'

O

-i i i -~ p h ~ . O

O

,,,, BzO

2.1.4 R=R'=TES ~ i 2.1.11 R=R'=H "h ii 2.1.12 R=H, R'=TES

2.1.13 R=TES "h Jr i 2.1.14 R=H

Conditions: (i) Py, 48%HF, CH3CN, 5 ~ 2.1.4 to 2.1.11, 79%; 2.1.13 to 2.1.14, 87%; (ii) TESC1, imidazole, DMF, 91%; (iii) LiHMDS, THF, 0 ~ then 2.1.10, 83%.

170 Under acidic conditions, a skeletal rearrangement of the A ring occurs, perhaps initiated by carbonium ion formation at C-1. A representative example is shown in Scheme 4. Kingston prepared ring-contracted paclitaxel analog 2.1.16 by reacting derivative 2.1.15 with methanesulfonyl chloride, followed by desilylation [21]. Later, similar rearrangements were observed by Potier [22] and Chen [23] under a variety of acidic conditions. In order to assess the contribution of the C-2 benzoate moiety to binding, a selective procedure for debenzoylation at C-2 was needed. Kingston has described a number of protocols for deacylation reactions of baccatin III. Under basic conditions, C-10 is deacylated first and, in certain cases, even the C-4 acetate is more labile than the C-2 benzoate [24]. Special conditions are therefore needed for selective C-2 deacylation.

AcO ph/~~_

0

Bz.. NH 0

Bz" NH 0 0 ....

phiaL-

i "

2.1.15

oH

O,

III

-
200

HCT-116

27

2.1.26a

p-MeO-C6H4-

Ph

>20

HCT-116

27

2.1.26b

p-NO2-C6H4-

Ph

>100

HCT-116

27

2.1.26c

c-Hex

Ph

11

HCT-116

27

2.1.26c

c-Hex

Ph

56

P-388

31

2.1.26d

Me

Ph

>20

HCT-116

27

3.1.1

o-C1-C6H4-

Ph

0.01

P-388

29

3.1.2

m-C1-C6H4-

Ph

0.0014

P-388

29

3.1.3

p-C1-C6H4-

Ph

150

P-388

29

3.1.4

m-CN-C6H4-

Ph

0.33

P-388

29

3.1.5

m-N3-C6H4-

Ph

0.002

P-388

29

3.1.6

m-NH2-C6H4-

Ph

1,500

P-388

29

3.1.7

m-CF3-C6H4-

Ph

15

P-388

29

3.1.8

m-F-C6H4-

Ph

0.35

P-388

29

3.1.9

2-Furyl

Ph

25

UCLA-P3

84

3.1.10

2-Thienyl

Ph

4.2

UCLA-P3

84

3.1.11

2-Naphthyl

Ph

>1,000

UCLA-P3

84

3.1.12

c-Hex

t-BuO

1.1

B-16

64

3.1.13

c- H ex

t-BuO b

11

P-388

31

(a) Concentration of analog that inhibits cell proliferation by 50% divided by concentration of paclitaxel that achieves same result. (b) This compound is a 10-deacetyl derivative. These observations suggest t h a t the i n t a c t A-ring s u b u n i t is an i m p o r t a n t structural element for cytotoxicity. The C-2 benzoate clearly plays a role in the cytotoxicity of paclitaxel, since 2-deoxytaxol, 2.1.41, is essentially inactive [26].

237 Due to the important role of the C-2 substituent for proper binding, it is clear t h a t small modifications at this site may lead to optimization of the activity, and it is not surprising t h a t several groups have reported efforts in this direction (Table 1). Several conclusions may be derived from the results in the table, even though cytotoxicities are generally reported for different cell lines. First of all, introduction of p a r a substituent at the benzoate invariably leads to loss of activity (see 2.1.26a,b and 3.1.3). Thus, the fit of the benzoate within the binding site seems to be r a t h e r tight, unless complex conformational changes are engendered by the p a r a substitution. It is unclear whether an aromatic ester at C-2 is needed for activity. Both Chen et al. [27] and Ojima et al. [31] have shown t h a t reduction of the C-2 benzoate to a cyclohexanoate leads to substantial loss of activity (see 2.1.26c and 3.1.13). In contrast, Georg et al. [64], working in the 10-acetyltaxotere series (see 3.1.12), report no loss of activity on hydrogenation of the C-2 benzoate. These apparent discrepancies are very difficult to interpret. Smaller aliphatic C-2 esters [27] (see 3.1.26d) and heteroaromatic ones [84] (see 3.1.9 and 3.1.10) are also poorly active. The most productive modifications to date have been carried out by Kingston et al., who reported large increases in cytotoxicity for some C-2 oand m - s u b s t i t u t e d benzoates [29]. In addition to i m p r o v e m e n t s in the cytotoxicity vs. paclitaxel, these analogs are clearly more effective in promoting microtubule assembly in vitro. These compounds, especially 3.1.2 and 3.1.5, are therefore promising, although neither data vs. resistant cell lines nor i n vivo evaluation have been reported. 5.3.2. Paclitaxel Analogs Modified at C-4 Chen and co-workers have extensively explored the SAR at C-4. Table 2 shows some of the highlights. As with C-2, deacylation or deoxygenation at C-4 leads to complete loss of activity (see 2.2.16 and 2.2.45). Introduction of large groups is also deleterious (2.2.24h), but aliphatic esters slightly larger than acetyl lead to improved activity (2.2.24j). Small carbonates and carbamates at C-4 are also quite active, especially in conjunction with improved side chains (for details on improved side chains, see chapter 6).

238

a'~. NH

O

\

AcO

O OH

Iii1< OH HO

BzO

X

Table 2: Cytotoxicity of Paclitaxel Analogs Modified at C-4 Cpd.

R

R'

X

IC50/IC50

Ref.

(paclitaxel) a 2.2.45

Ph Ph

Bz Bz

OH H

>25 n.d. b

19, 32 36

2.2.24h

Ph

Bz

OBz

100

20

2.2.28a

Ph

Bz

OCO2Me

0.9

19

2.2.24j

Ph

Bz

OCO-c-Pr

0.4

20

3.2.1

Ph

Bz

3.9

19

4.5 d

35

2.2.1{}

Ok\ 2 - - N-~J ~

l-o 3.2.2

t- oc

O N.

l-o / - - 3.2.3

Ph

Bz c

",,1

OCO-i-Pr

(a) Measured in HCT-116 cells. (b) Very poor tubulin polymerization activity. (c) This compound is a 10-deacetyl derivative. (d) Measured in B-16 melanoma cells. Georg et al. have also reported a derivative modified at C-4, 3.2.3, which is slightly less active t h a n paclitaxel. Upon further exploration, modification of the C-4 function is likely to afford very potent derivatives. 5.3.3. Paclitaxel Analogs Modified at the Oxetane Ring Many of the derivatives described in section 5.2.3, in which the oxetane ring has been opened, were evaluated for their ability to polymerize tubulin [23]. None of t h e m displayed any measurable activity and they are therefore useless in defining the SAR at this locus. Most of them are missing an acetoxy group at C-4 which, as we discussed above, is crucial for binding. This is in agreement with Kingston's early results [2]. Thus, the oxetane seems to play

239 an essential role in the binding of paclitaxel to microtubules. It is not known whether the oxetane acts to rigidify ring C and point the C-4 acetoxy group in the appropriate direction for binding, or whether the oxetane oxygen itself is a binding element. The derivatives that would answer this question (e.g. the one bearing a cyclobutane ring in place of the oxetane) have not yet been described. 5.3.4. Analogs Modified at the C-7 Position C-7 Xylosyltaxol, 3.4.1 was isolated from the bark and leaves of T a x u s b a c c a t a , and shown to be more potent t h a n paclitaxel in the tubulin polymerization assay [3, 85]. Other analogs were prepared either from baccatin derivatives or from paclitaxel itself. Most of the analogs tested (Table 3) have in vitro activity comparable to paclitaxel and docetaxel. With the exception of large lipophilic substituents such as the silyl ethers (see 3.4.8 and 3.4.10), any modification seems well tolerated, with some analogs being slightly more cytotoxic than paclitaxel. It seems likely that the C-7 substituent is not engaged in significant interactions at the binding site, and t h a t chemical modification at this position will only serve to m o d u l a t e the activity (perhaps via altered solubility, metabolism, biodistribution). Some of the C-7 derivatives were tested in vivo. With the exception of the carbonate derivative 3.4.6, none of the

compounds compared favorably with paclitaxel at their MTD. Interestingly, even cyclopropane derivative 2.4.14 retains in vitro activity, in spite of the slight conformational alteration imparted to the C ring vs. paclitaxel. 5.3.5. Analozs Modified at the C-9 Carbonyl This section examines primarily the effect of reducing the C-9 carbonyl on the cytotoxicity. Some analogs carry also C-10 and/or C-7 modifications and are discussed here for the sake of convenience. As with the C-7 position, even with the small database available, it is clear t h a t most modifications, including complete defunctionalization, are well tolerated at C-9. None of the modifications effected led to complete loss of activity. The derivative 2.5.31, f e a t u r i n g a completely defunctionalized northern half, is only 5-6 times less active than paclitaxel. Compounds with a partially hydroxylated northern segment have activities of the same level as paclitaxel and docetaxel (Table 4).

240

R "NH O

AcO

O X

p h - - " L ~ _ O .... OH HO

" BzO

Table 3: Cytotoxicity of Paclitaxel Analogs Modified at C-7 Cpd.

R

X

In vitro

Cell

In vivo

IC50/IC50

Line

activityb

Ref.

paclitaxel a 3.4.1

Bz

([~)Xylosyl

n.d.c

-

3

3.4.2

Bz

([~)OAc

1.3

J774.2

85

3.4.3

Boc

(~)L-Phenyl

0.44

P-388

52

3.4.4

Bz

([~) L-Alanyl

2.3

B-16

86

3.4.5

Bz

([~)N,N-dimethyl

2.3

S-16

86

3.4.6

Bz

(~)OC02Et

3.4.7

Bz

3.4.8

Bz

3.4.8 2.4.1

alanyl d

glutaryl 1.5

HCT-116

289 (40)

([~)OCONHBu

1.0

HCT-116

157 (50)

([3)OMs

0.9

HCT-116

Bz

([~)OSiEt3

>20

HCT-116

Bz

(a)OH

0.5

HCT-116 126-154 (30-

-

-

45 45 45 45 45

32) 3.4.10

Bz

(a)OSiMe3

>20

HCT-116

2.4.7

Bz

2.4.8

Boc

H

1.0

HCT-116

157 (50)

45

H

0.4

HCT-116

156 (64)

45

2.4.16

Bz

2.4.14

Bz

A6-dehydro

1.2

HCT-116

161 (60)

45

A7,19-cyclopropa

2.0

HCT-116

2.4.12

Bz

156 (80)

45

(a)F

2.9

HCT-116

185 (132)

45

3.4.11

Boc

(a)F

1.2

HCT-116

147 (40)

45

-

45

(a) See Table 1 for definition. (b) I n v i v o data in unstaged M109 model. Values indicate T/C at the MTD (mg/Kg/inj., in parentheses). Paclitaxel gave T/C values of 183-276 at MTDs of 50-75 mg/Kg/inj. (c) IC50 0.4 v s . paclitaxel (1.0) in tubulin polymerization assay. (d) A C-10 deacetyl derivative.

241

R "NH 0 ph-~,~_

X

Y Z

0 ....

OH

0 HO

" BzO

OAc

Table 4: Paclitaxel Analogs Modified at the C-9 Carbonyl Cpd.

R

X

Y

Z

IC5o/IC5o (paclitaxel)a

Cell Line

Ref.

2.5.1

Boc

(~)OH

(a)OH

2.5.2

Boc

( [ 3 ) O H (~)OH

(~)OH

1.9b

P-388

56

([~)OH

2.0b

P-388

56

2.5.4

Boc

(~)OH

(a)OH

(a)OH

3.2b

P-388

56

2.5.10

Bz

3.5.1

Bz

([~)OAc

(a)OH

(~)OH

8-10

P-388

17

([~)OAc

H

(~)OH

0.5

P-388

17

3.5.2

Bz

([~)OAc

H

H

1

P-388

17

2.5.31 2.5.24

Bz Bz

H H

H (~)OH

H (~)OH

5-6 14

P-388 B-16

17 64

2.5.25

Boc

(~)OH (~)OH

B-16

64

Boc

(~)OH (~)OH

1.1

2.5.26

(~)OH H

1.8

B-16

64

(a) See Table 1. (b) This value is referenced to docetaxel, not paclitaxel. Reduction of the C-9 carbonyl yields active a or 13carbinols (see 2.5.1 and 2.5.2). A 10-deoxy-9-dihydroderivative is much less bioactive t h a n paclitaxel, b u t simply switching the side chain to the one from docetaxel restores the activity (see 2.5.24

vs.

2.5.26). These observations reinforce the notion t h a t the northern

half of the molecule does not intimately interact with the microtubule binding site. 5.3.6. Analogs Modified at the C-10 Position Table 5 shows some of the analogs t h a t bear modified C-10 substituents. Some also bear modifications at C-7 and are discussed here for the sake of convenience. Although it is well known t h a t introduction of polar esters or other functions at C-10 leads to loss of activity [52], minor modification with

242 relatively small substituents at this position have been shown to lead to active analogs [66].

X RHN

0

P h i _ _-

Oy

\

0 ....
C-18 [60]. C-12 fluorinated derivatives, where the double bond has moved into conjugation with the C-9 carbonyl, were also obtained as side products. Biological evaluation of some of these compounds (see Figure 3) shows that migration of the C-11/C-12 double bond leads to some loss in activity. Ten-fold drops in cytotoxicity (vs. paclitaxel) are seen with dienones 2.5.18, 3.7.1 and 3.7.2. The fluorinated derivatives are also ten-fold less active than paclitaxel, except for derivative 3.7.3, which bears a ~-methyl grouop at C-12, and is over 100-fold less active [19]. It is likely that, due to the importance of the C-13 side chain in the binding process, its exact spatial positioning is crucial to the activity of these analogs. Even slight conformational changes in the A ring might simply alter the spatial relationship of the side chain vs. the other binding elements in the molecule (the C-2 and C-4 esters). 5.3.8. Analogs Modified at the C-14 Position The biological activity of analogs bearing a functionalized C-14 has been explored in a preliminary fashion by Kant et al. [12] and Ojima et al. [13, 70]. The 14-(~)OH analog of paclitaxel, 2.7.14 (Table 6) shows slightly reduced cytotoxicity vs. the parent drug. I n v i v o evaluation showed that this derivative is essentially devoid of antitumor activity [12]. Switching the side chain to the one found in docetaxel, as predicted, results in a slightly improved performance (see 2.7.15 and 2.7.11) [12]. Even a cyclic carbonate at C-14/C-1 is compatible with good activity, but only in the presence of the docetaxel side chain, the paclitaxel analog being remarkably less active (2.7.12 vs. 3.8.1). A C-1/C-14 acetonide (see 2.7.13) is deleterious to activity.

244

#

BzHN O

O

R

O ....~ OH

2.5.18 3.7.1 3.7.2

R=(~)OH R=(~)OCOCHFC1 R=(cz)OH

ICso/ICso(pacl) 9.5 9.5 10 O

BzHN O RI,,,,~..~ ~ , ~ o ....

Z'.. i

BzO 3.7.3 3.7.4 3.7.5

J,

OAc

RI= F; R2 =Me; 1~3 = (a)OH RI= Me; R2 =F; R3 = (a)OH RI= Me; R2 =F; R3 = (~)OH

Figure 3: Paclitaxel analogs modified at C-10/C-12 and their

IC5o/IC5o(pacl) 230 13.5 9.5 in vitro

cytotoxicity (HCT-116)

Analogs where the side chain was introduced at C-14 instead of C-13 were much less active than docetaxel [13, 70]. Not enough is known about the SAR at C-14 to draw final conclusions as to the involvement of this position at the binding site. Since most of the analogs in Table 6 have similar activity to paclitaxel, it is likely that the C-14 functionality does not perform a binding function, and therefore only minor changes in cytotoxicity can be realized by fine-tuning such functionality. 5.3.9. Misce!!.aneous Analogs Klein reported on the synthesis of novel paclitaxel derivatives featuring a contracted seven-membered B-ring, 3.9.1 and 3.9.2 (Figure 4 ) [ 4 8 ] . Interestingly, these compounds were of comparable activity to paclitaxel in the

245 in vitro P-388 cytotoxicity assay. More work needs to be done to assess the

potential of these unusual analogs.

FI~HN

PhA

O

\

2

OH

_- Lo .... OH

_ R40 OR 3 0 i =

O

Table 6: Cytotoxicity of Derivatives Modified at C-14. Cpd.

R1

R2

R3,R4

IC5o/IC50 paclitaxel a

Cell Line

Ref.

2.7.14

Bz

Ac

H,H

4.0

HCT-116

12

2.7.15 2.7.11 2.7.12

Boc Boc Boc

Ac H H

3.8.1

Bz

H

2.7.13

Boc

H

H,H H,H C=O C=O C(Me)2

1.0 1.0 1.0 17 7.5

HCT-116 HCT-116 A121 A121 A121

12 12 13, 70 13, 70 13, 70

(a) See Table 1. The Ojima group described the synthesis and biological evaluation of two novel nor-seco analogs of paclitaxel and docetaxel, 2.8.44 and 2.8.45 [74]. These compounds are 20-40-fold less potent than paclitaxel in a number of tumor cell lines. These results thus clearly indicate the importance of the A-ring for the proper binding of paclitaxel and docetaxel to their biological target. A pentacyclic paclitaxel derivatives (2.9.4), prepared by photochemical irradiation of paclitaxel by Chen et al. [76], failed to show any activity in the tubulin polymerization assay as well as in cytotoxicity assays. The core of this molecule is grossly distorted with respect to the one in paclitaxel, and no activity would be expected. A recent report from Commerqon et al. provides the first example of a C19 modification [88]. The fact that a C-19-hydroxylated docetaxel analog (3.9.3) exhibits slightly better activity than the parent drug in the tubulin disassembly

246 assay suggests that chemical modifications at C-19 may lead to useful derivatives. AcO R.N o ,, X,o.c o. 3.9.1 R=Bz 3.9.2 R=Boc OBz OAc RHN

O

0 _. // OH

\

~ ~OHo

,

O .

HO

BzHN "

Ogz OAc

AcO I ~, / H,,,

0

O

/? OH \l~ 2.9.4

o ....

HO

dR HO

BocHN

O

P h ~ O

~

/

O/OH

~ 3.9.3

~

~-~o

HO BzHN

OAc

....

o.

0

OBz OAc

AcO

O

I~,,,,,II

jOH ~oH'" 3.9.4

p h ~ - ~ ' O _ ....

o.

2.8.44 R=Bz 2.8.45 R=Boc

~ HO

~~o (DBz OAc

Figure 4: Miscellaneous paclitaxel and docetaxel derivatives

247 Finally, among the many docetaxel metabolites isolated, one (3.9.4) features a novel core functionalization, i.e. a hydroxyl group at C-6. Such hydroxylation leads to a 30-fold drop in activity vs. docetaxel, i.e. detoxification of the drug [89]. 5.3.10. Conclusion Although much work remains to be done in this area, a qualitative picture of the SAR of paclitaxel is beginning to emerge. At least three functional elements, i.e. the C-13 side chain (see chapter 6) and the C-2 and C-4 esters, are intimately involved in interactions at the binding site. It appears that the northern half of the molecule and the tetracyclic skeleton (including an intact oxetane) function essentially as a molecular scaffolding to hold these binding elements in the proper orientation. Some uncertainty still exists about a possible binding role for the oxetane oxygen and the C-1 hydroxyl group. Modifications of the essential functions may therefore lead (and in some cases this has been achieved in cell culture) to more potent paclitaxel analogs, through further optimization of the fit with the microtubule site, whereas modifications at the non-essential positions may modulate the activity by changing the physico-chemical parameters of the molecule or via other secondary effects. R~'EgENCIi~

.

.

.

.

.

7.

Wani, M.C.; Taylor H.L.; Wall, M.E.; Coggon, P.; McPhail, A . T . J . Am. Chem. Soc. 1971, 93, 2325. Kingston, D.G.I.; Samaranayake, G.; Ivey, C.A.J. Nat. Prod. 1990, 53, 1. S6nilh, V.; Blechert, S.; Colin, M.; Gu6nard, D.; Picot, F.; Potier, P.; Varenne, P. J. Nat. Prod. 1984, 47, 131. Denis, J.N.; Greene, A.E.; Gu6nard, D.; Gu6ritte-Voegelein, F.; Mangatal, L.; Potier, P. J. Am. Chem. Soc. 1988, 110, 5917. Mangatal, L.; Adeline, M.T.; Gu6nard, D.; Gu6ritte-Voegelein, F.; Potier, P. Tetrahedron 1989, 45, 4177. Rowinsky, E.R.; Donehower, R.C. Pharmacol. Ther. 1991, 52, 35. For recent reviews dealing with chemistry and structure-activity relationship studies, see: (a) Kingston, D.G.I. Pharmacol. Ther. 1991,

248 52, 1. (b) Kingston, D.G.I.; Molinero, A.A.; Rimoldi, J.M.; In: Progress in the Chemistry of Organic Natural Products; Herz, W.; Kirby, G.W.;

o

.

10.

11. 12. 13. 14. 15. 16.

17.

Moore, R.E.; Steglich, W.; Tamm, Ch., Eds; Springel-Verlag, New York, 1993; vol. 61, p.1. (c) Gu~nard, D.; Gu~ritte-Voegelein, F.; Potier, P. Acc. Chem. Res. 1993, 26, 160. (d) Georg, G.I.; Boge, T.C.; Cheruvallath, Z.S.; Clowers, J.S.; Harriman, G.C.B.; Hepperle, M.; Park, H. In: Taxol: Science and Applications, Suffness, M., Ed.; CRC, Boca Raton, in press. (e) Nicolaou, K.C.; Dai, W.M.; Guy, R.K. Angew. Chem. Int. Ed. Engl. 1994, 33, 15. Reviews: (a) Swindell, C.S. Org. Prep. Proced. Int. 1991, 23,465. (b) Blechert, S.;Gu~nard, D.In:The Alkaloids, Chemistry and Pharmacology, Brossi, A., Ed.; Academic Press, San Diego, 1990; Vol. 39, p.195. (c) Paquette, L.A. In: Studies in Natural Product Chemistry; Atta-ur-Rahman, Ed.; Elsevier, 1992; Vol.ll, p.3. Holton, R.A.; Somoza, C.; Kim, H.B.; Liang, F.; Biediger, R.J.; Boatman, P.D.; Shido, M.; Smith, C.C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, K.K.; Gentile, L.N.; Liu, J . H . J . Am. Chem. Soc. 1994, 116, 1597; 1599. Nicolaou, K.C.; Yang, Z.; Liu, J.J.; Ueno, H.; Nantermet, P.G.; Guy, R.K.; Claiborne, C.F.; Renaud, J.; Couladouros, E.A.; Paulvannan, K.; Sorensen, E.J. Nature 1994, 367, 630. Klein, L.L. Tetrahedron Lett. 1993, 34, 2047. Kant, J.; Farina, V.; Fairchild, C.; Kadow, J.F.; Langley, D.R.; Long, B.H.; Rose, W.C.; Vyas, D.M. Bioorg. Med. Chem. Lett. 1994, 4, 1565. Ojima, I.; Fenoglio, I.; Park. Y.H.; Pera, P.; Bernacki, R.J. Bioorg. Med. Chem. Lett. 1994, 4, 1571. Suffness, M. Ann. Rep. Med. Chem. 1993, 28, 305. Klein, L.L. Presented at the 207th Meeting of the American Chemical Society, San Diego, 1994. Abstract #MEDI143. (a) Chen, S.H.; Huang, S.; Gao, Q.; Golik, J.; Farina, V. J. Org. Chem. 1994, 59, 1475. (b) Chen, S.H.; Huang, S., submitted for publication. (c) Chaudhary, A.G.; Chordia, M.D.; Kingston, D.G.I., submitted for publication. Klein, L.L.; Li, L.; Yeung, C.M.; Mating, C.J.; Thomas, S.A.; Grampovnik, D.J.; Plattner, J.J. In: Taxane Anticancer Agents: Basic

249 Science and Current Status; Georg, G.I.; Chen, T.T.; Ojima, I.; Vyas,

18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

28. 29.

30. 31. 32. 33.

D.M., Eds.; ACS Symposium Series 583, Washington (1995), p.276. Holton, R.A. US Patent 5, 015, 744 (1991); US Patent 5, 136,060 (1992). Chen S.H., unpublished results. Chen, S.H.; Kadow, J.F.; Farina, V.; Johnston, K.; Fairchild, C. J. Org. Chem. 1994, 59, 6156. Samaranayake, G.; Magri, N.; Jitrangsri, C.; Kingston, D.G.I.J. Org. Chem. 1991, 56, 5114. Wahl, A.; Gu~ritte-Voegelein, F.; Gu~nard, D.; Le Goff, M.T.; Potier, P. Tetrahedron 1992, 48, 6965. Chen, S.H.; Huang, S.; Wei, J.M.; Farina, V. Tetrahedron 1993, 49, 2805. Samaranayake, G.; Neidigh, K.A.; Kingston, D.G.I.J. Nat. Prod. 1993, 56, 884. Farina, V.; Huang, S. Tetrahedron Lett. 1992, 33, 3979. Chen, S.H.; Wei, J.M.; Farina, V. Tetrahedron Lett. 1993, 34, 3205. (a) Chen, S.H.; Farina, V.; Wei, J.M.; Long, B.H.; Fairchild, C.; Mamber, S.; Kadow, J.F.; Vyas, D.M.; Doyle, T.W. Bioorg. Med. Chem. Lett. 1994, 4, 479. (b) Gao, Q.; Wei, J.M.; Chen, S.H. Pharm. Res., in press. Datta, A.; Jayasinghe, L.R.; Georg, G.I.J. Org. Chem. 1994, 59, 4689. Chaudhary, A.; Gharpure, M.D.; Rimoldi, J.M.; Chordia, M.D.; Gunatilaka, A.A.L.; Kingston, D.G.I.; Grover, S.; Lin, C.M.; Hamel, E. J. Am. Chem. Soc. 1994, 116, 4097. Nicolaou, K.C.; Nantermet, P.G.; Ueno, H.; Guy, R.K.J. Chem. Soc. Chem. Commun. 1994, 295. Ojima, I.; Duclos, O.; Zucco, M.; Bissery, M.C.; Combeau, C.; Vrignaud, P.; Riou, J.F.; Lavelle, F. J. Med. Chem. 1994, 37, 2602. Neidigh, K.A.; Gharpure, M.M.; Rimoldi, J.M.; Kingston, D.G.I.; Jiang, Y.Q.; Hamel, E. Tetrahedron Lett. 1994, 35, 6839. Gu~ritte-Voegelein, F.; Gu~nard, D.; Dubois, J.; Wahl, A.; Marder, R.; Muller, R.; Lund, M.; Bricard, L.; Potier, P. In: Taxane Anticancer Agents: Basic Science and Current Status; Georg, G.I.; Chen, T.T.; Ojima, I.; Vyas, D.M., Eds.; ACS Symposium Series 583, Washington (1995), p.276.

250 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

46. 47. 48.

49. 50. 51. 52.

Chen, S.H.; Long, B.H.; Fairchild, C. J. Org. Chem. submitted for publication. Georg, G.I.; Ali, S.M.; Boge, T.C.; Datta, A.; Falborg, L.; Himes, R.H. Tetrahedron Lett. 1994, 35, 8931. Chordia, M.D.; Chaudhary, A.G.; Kingston, D.G.I.; Jiang, Y.Q.; Hamel, E. Tetrahedron Lett. 1994, 35, 6843. Gu6ritte-Voegelein, F.; Gudnard, D.; Potier, P. J. Nat. Prod. 1990, 50, 9. Huang, C.H.O.; Kingston, D.G.I.; Samaranayake, G.; Boettner, F . E . J . Nat. Prod. 1986, 49, 665. Magri, N.F. Ph.D. Dissertation, Virginia Polytechnic Institute and State University, 1985. Chaudhary, A.G.; Rimoldi, J.M.; Kingston, D.G.I.J. Org. Chem. 1993, 58, 3798. Chen, S.H.; Huang, S.; Kant, J.; Fairchild, C.; Wei, J.M.; Farina, V. J. Org. Chem. 1993, 58, 5028. Chen, S.H.; Huang, S.; Farina, V. Tetrahedron Lett. 1994, 35, 41. Chen, S.H.; Gao, Q. unpublished results. Roth, G.P.; Marshall, D.; Chen, S.H. submitted for publication. Chen, S.H.; Kant, J.; Mamber, S.W.; Roth, G.P.; Wei, J.M.; Vyas, D.M.; Farina, V.; Casazza, A.; Long, B.H.; Rose, W.C.; Johnston, K.; Fairchild, C. Bioorg. Med. Chem. Lett. 1994, 4, 2223. Johnson, R.A.; Nidy, E.G.; Dobrowolski, P.J.; Gebhard, I.; Qualls, S.J.; Wicnienski, N.A.; Kelly, R.C. Tetrahedron Lett. 1994, 35, 7893. Chen, S.H.; Huang, S.; Wei, J.M.; Farina, V. J. Org. Chem. 1993, 58, 4520. Klein, L.L.; Maring, C.J.; Li, L.; Yeung, C.M.; Thomas, S.A.; Grampovnik, D.J.; Plattner, J.J.; Henry, R.F.J. Org. Chem. 1994, 59, 2370. Magri, N.F.; Kingston, D.G.I.J. Org. Chem. 1986, 51,797. Mellado, W.; Magri, N.F.; Kingston, D.G.I.; Garcia-Arenas, R.; Orr, G.A.; Horwitz, S.B. Biochem. Biophys. Res. Commun. 1984, 124, 329. Magri, N.F.; Kingston, D.G.I.; Jitrangsri, C.; Piccariello, T. J. Org. Chem. 1986, 51, 3239. Gu6ritte-Voegelein, F.; Gu~nard, D.; Lavelle, F.; LeGoff, M.T.; Mangatal, L.; Potier, P. J. Med. Chem. 1991, 34, 992.

251 53.

54. 55. 56. 57.

58. 59. 60. 61. 62. 63. 64.

65. 66.

67. 68.

Deutsch, H.M.; Glinski, J.A.; Hernandez, M.; Haugwitz, R.D.; Narayanan, V.L.; Suffness, M.; Zalkow, L.H.J. Med. Chem. 1989, 32, 788. Mathew, A.; Mejillano, M.R.; Nath, J.P.; Himes, R.H.; Stella, V. J. Med. Chem. 1991, 35, 145. Magri, N.F.; Kingston, D.G.I.J. Nat. Prod. 1988, 51,298. Pulicani, J.P.; Bourzat, J.-D.; Bouchard, H.; Commerqon, A. Tetrahedron Lett. 1994, 35, 4999. (a) Gunawardana, G.P.; Premachandran, U.; Burres, N.S.; Whittern, D.N.; Henry, R.; Spanton, S.; McAlpine, J . B . J . Nat. Prod. 1992, 55, 1686. (b) Zamir, L.O.; Nedea, M.E.; Belair, S.; Sauriol, F.; Mamer, O.; Jacqmain, E.; Jean, F.I.; Garneau, F.X. Tetrahedron Lett. 1992, 33, 5173. Maring, C.J.; Grampovnik, D.J.; Yeung, C.M.; Klein, L.L.; Li, L.; Thomas, S.A.; Plattner, J.J. Bioorg. Med. Chem. Lett. 1994, 4, 1429. Datta, A.; Aub~, J.; Georg, G.I.; Mitscher, L.A.; Jayasinghe, L.R. Bioorg. Med. Chem. Lett. 1994, 4, 1831. Chen, S.H.; Fairchild, C.; Mamber, S.W.; Farina, V. J. Org. Chem. 1993, 58, 2927. Chaudhary, A.G.; Kingston, D.G.I. Tetrahedron Lett. 1993, 34, 4921. Chen, S.H.; Huang, S.H.; Vyas, D.M.; Doyle, T.W.; Farina, V. Tetrahedron Lett. 1993, 34, 6845. Holton, R.A.; Somoza, C.; Chai, K.B. Tetrahedron Lett. 1994, 35, 1665. Georg, G.I.; Harriman, G.C.B.; Vander Velde, D.G.; Boge, T.C.; Cheruvallath, Z.S.; Datta, A.; Hepperle, M.; Park, H.; Himes, R.H.; Jayasinghe, L. In: Taxane Anticancer Agents: Basic Science and Current Status; Georg, G.I.; Chen, T.T.; Ojima, I.; Vyas, D.M., Eds.; ACS Symposium Series 583, Washington (1995), p.217. Georg, G.I.; Cheruvallath, Z.S.J. Org. Chem. 1994, 59, 4015. Kant, J.; O'Keeffe, W.S.; Chen, S.H.; Farina, V.; Fairchild, C.; Johnston, K.; Kadow, J.; Long, B.H.; Vyas, D.M. Tetrahedron Lett. 1994, 35, 5543. Gu~ritte-Voegelein, F.; S~nilh, V.; David, B.; Gu~nard, D.; Potier, P. Tetrahedron 1986, 42,4451. Taylor, G.F.; Thornton, S.C.; Tallent, C.R.; Kepler, J . A . J . Labelled Cpds. and Radiopharm. 1993, 33, 501.

252 69. 70. 71. 72.

73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87.

Appendino, G.; Gariboldi, P.; Gabetta, B.; Pace, R.; Bombardelli, E.; Viterbo, D. J. Chem. Soc. Perkin Trans. 1 1992, 2925. Ojima, I.; Park, Y.H.; Sun, C.M.; Fenoglio, I.; Appendino, G.; Pera, P.; Bernacki, R.J.J. Med. Chem. 1994, 37, 1408. Py, S.; Khuong-Huu, F. Bull. Soc. Chim. Fr. 1993, 130, 189. (a) Appendino, G.; Varese, M.; Gariboldi, P.; Gabetta, B. Tetrahedron Lett. 1994, 35, 2217. (b) Appendino, G.; Cravotto, G.; Enrifi, R.; Jakupovic, J.; Gariboldi, P.; Gabetta, B.; Bombardelli, E. Phytochemistry 1994, 36, 407. Appendino, G.; Jakupovic, J.; Cravotto, G.; Varese, M. Tetrahedron Lett. 1994, 35, 6547. Ojima, I.; Fenoglio, I.; Park, Y.H.; Sun, C.M.; Appendino, G.; Pera, P.; Bernacki, R. J. Org. Chem. 1994, 59, 515. Py, S.; Pan, J.W.; Khuong-Huu, F. Tetrahedron 1994, 50, 6881. Chen, S.H.; Combs, C.M.; Hill, S.E.; Farina, V.; Doyle, T.W. Tetrahedron Lett. 1992, 33, 7679. Chiang, H.C.; Wood, M.C.; Nakanaira, Y.; Nakanishi, K. J. Chem. Soc., Chem. Commun. 1967, 1201. Chen, S.H.; Farina, V.; Huang, S.; Gao, Q.; Golik, J.; Doyle, T.W. Tetrahedron 1994, 50, 8633. Swindell, C.S.; Krauss, N.E.; Horwitz, S.B.; Ringel, I. J. Med. Chem. 1991, 34, 1176. Georg, G.I.; Cheruvallath, Z.S.; Himes, R.H.; Mejillano, M.R.; Burke, C.T.J. Med. Chem. 1992, 35, 4230. Long, B.H. unpublished results. Suffness, M. Ann. Rep. Med. Chem. 1993, 305. Rose, W.C. Anti-Cancer Drugs 1992, 3, 311. Nicolaou, K.C.; Couladouros, E.A.; Nantermet, P.G.; Renaud, J.; Guy, R.K.; Wrasidlo, W. Angew. Chem. Int. Ed. Engl. 1994, 33, 1581. Lataste, H.; S~nilh, V.; Wright, M.; Gu~nard, D.; Potier, P. Proc. Natl. Acad. Sci. USA 1984, 81, 4090. Mathew, A.E.; Mejillano, M.R.; Nath, J.P.; Himes, R.H.; Stella, V. J. Med. Chem. 1992, 35, 145. Klein, L.L.; Yeung, C.M.; Li, L.; Plattner, J.J. Tetrahedron Lett. 1994, 35, 4707.

253 88. 89.

Margraff, R.; B6zard, D.; Bourzat, J.D.; Commer~on, A. Bioorg. Med. Chem. Lett. 1994, 4, 233. Harris, J.W.; Katki, A.; Anderson, L.W.; Chmurny, G.N.; Paukstelis, J.V.; Collins, J.M.J. Med. Chem. 1994, 37, 706.

The Chemistry and Pharmacology of Taxol and its Derivatives V. Farina, editor 9 1995 Elsevier Science B. V. All rights reserved

255

6 THE C H E M I S T R Y OF THE TAXOL| S I D E CttAIN: S Y N T H E S I S , MODIFICATIONS AND CONFORNATIONAL STUDIES Joydeep Kant Johnson Matthey Inc., Biomedical Materials, 2003 Nolte Drive, West Deptford, New Jersey 08066, U.S.A.

6.1. INTRODUCTION

Taxol | (1.1.1), a complex antineoplastic diterpene isolated from Taxus brevifolia by Wani and Wall [1], has recently been approved for the treatment of cisplatin-refractory ovarian cancer and metastatic breast cancer [2,3]. The cytotoxicity of this drug is due to microtubule-mediated interruption of mitosis, which occurs through tubulin polymerization and formation of extremely stable and non-functional microtubules, which are abnormally resistant to depolymerization [4]. The limited availability of Taxol | from natural resources and its high clinical importance have stimulated considerable interest, within the synthetic community, toward the synthesis of this complex molecule and its analogs, with the aim of designing better antitumor drugs. Over the years, many approaches to the total synthesis of Taxol | have appeared in the l i t e r a t u r e [5], and recently Holton and Nicolaou, independently, have recorded successful approaches to this challenging target [6, 7]. Due to the length of these approaches, the total synthesis of Taxol| may not be feasible on an industrial scale. Taxol | was initially produced

256 exclusively by a tedious extraction procedure from the bark of the Pacific Yew; one Kg of the drug is isolated from the bark of 3,000 yew trees [8,9]. 0 18

AcO 0 ~ . _~19 OH

~3\

'2' ',,16,~ 4

'4

~

~'0

0

1.1.1 Taxol |

To circumvent this problem, Greene, Potier and coworkers [10] developed an efficient semi-synthetic approach. The chemistry involves an enantioselective synthesis of (2'R, 3'S)a-hydroxy-~-amino acid derivative 1.1.2 and its coupling to suitably protected 10-desacetylbaccatin III (10-DAB), 1.1.3, at the C-13 position. To date, the semi-synthetic approach appears to be the most practical way of producing Taxol| a large scale. Since 10-DAB is isolated from the needles of the widely distributed T a x u s b a c c a t a (yield: ca. lg/Kg dry leaves), a renewable source of 10-DAB is available in large quantities [11]. O

OH

Ph" ~ NH

O

- R oH 1.1.2

0

HO . . . . .

,,

n No 1.1.3

Thus, the success of the semi-synthetic approach relies on an efficient asymmetric synthesis of 1.1.2, followed by its attachment to 1.1.3. Therefore, in recent years, various academic and pharmaceutical research groups have

257 focused their efforts on the development of practical asymmetric syntheses of the side chain and its attachment to the baccatin core. Furthermore, these strategies have allowed the synthesis of a variety of side-chain analogs of Taxol| in this way helping to establish Structure-Activity Relationships (SAR) in search of more effective anti-tumor drugs. Since the discovery of Taxol| many reviews and accounts have appeared describing its chemistry and biology [5,12-19]. This chapter focuses only on the chemistry of the side chain. The first section describes a variety of chiral and non-chiral approaches to the synthesis of 1.1.2 and its mode of attachment to 1.1.3. The second section deals with the chemistry and biology of the side-chain modified analogs and their SAR. The final section summarizes what is known about the conformation of the Taxol | side chain in relation to the core and the biological mode of action. 6.2. SYNTHESES OF THE SIDE CHAIN 6.2.1. Asymmetric Epoxidation/Dihydroxylation Approaches Greene and co-workers employed the Sharpless asymmetric epoxidation followed by regioselective ring opening of the chiral epoxide 2.1.2 in their synthesis of the Taxol| side chain [20]. The key intermediate, 2.1.2 was prepared by Sharpless epoxidation from cis-cinnamyl alcohol 2.1.1, followed by oxidation of the epoxy alcohol and esterification of the resulting acid. The regiospecific ring opening of 2.1.2 using trimethylsilyl azide and zinc chloride afforded a - h y d r o x y - ~ - a z i d o amino acid 2.1.3 in high yield. Next, Obenzoylation followed by reduction produced the desired 2.1.5 via O->N-benzoyl migration. Although the epoxy alcohol was isolated with an enantiomeric excess (e.e.) of only 78%, a single recrystallization of 2.1.5 from chloroform afforded pure material in >95% e.e. (Scheme 1). The above chemistry was significantly improved by replacing the epoxidation step with the Sharpless dihydroxylation reaction. Treatment of i n e x p e n s i v e m e t h y l c i n n a m a t e , 2 . 1 . 6 , in aqueous acetone with dihydroquinidine-4-chlorobenzoate (DQCB) and N-methylmorpholine N-oxide (NMMO) in conjunction with a catalytic amount of OsO4, produced the diol 2.1.7 in fair yield with an e.e. of >98% [21]. Chemoselective tosylation at C-2 followed by mild base treatment afforded epoxide 2.1.2 which was converted to

258 2.1.5 in a one-pot procedure. Similarly, the side chain of Taxotere | 2.1.9, an

analog of Taxol |

was also synthesized (Scheme 2).

[Scheme 1]l Ph CH2OH \--/

i, i i, i i i

iv

=

2.1.2

oyp.

~

OMe OH 2.1.3

0

vi

O

phil_

Ph/JJxN H

N3 O II P h ~ O M e

=

~

O2Me

2.1.1

v

N3

0 ph , ~ ~ ~

0

phil"_

OMe

OH

0

2.1.5

2.1.4

Conditions: (i) t-BuO2H, Ti(OiPr)4, L-diethyl tartrate, CH2C12, -30 ~ 61%; (ii) RuC13, NaIO4, NaHCO3, CC14, CH3CN/H20; (iii) CH2N2, ether, 84% overall; (iv) TMSN3, ZnC12, H3O+, 90%; (v) PhCOC1, NEt3, DMAP, CH2C12, 94%; (vi) H2, PcYC,MeOH, then Ar, 89% (>95% e.e.).

Scheme 21 OH Ph@co2Me

i

0

~ Ph

0 OMe

=

p

O2Me

OR

2.1.6

/" 2.1.7 R = H ii ~ 2.1.8 R = OTs N3

iv

=

~ Ph

2.1.2 O R'~NH

O

O

v

OH

2.1.3

OMe

or v i

ph/~~_

OMe

OH

2.1.5 R=Ph 2.1.9 R=tBuO

(i) DQCB, NMMO, Os04 (cat.), acetone/H20, 51% (>98% e.e.); (ii)p-TsC1, NEt3, CH2C12, 88%; (iii) K2C03, H20, DMF, 91%; (iv) NAN3, AcOH, MeOH/H20, 95%; (v) PhCOC1, NEt3, DMAP; H2, Pd/C, EtOAc, 92%; (vi) Boc20, H2, Pd/C, EtOAc, 92%. Conditions:

259 Coupling of 2.1.5 to baccatin was initially reported to be a very difficult operation, probably due to the hindered nature of the C-13 hydroxyl group in baccatin [10]. The C-2 hydroxyl group in 2.1.5 was protected as an acid-labile ethoxyethyl ether and the ester was hydrolyzed to the free amino acid 2.1.10. Treatment of 7-triethylsilyl (TES) baccatin III (2.1.11) in toluene with 6 equiv of 2 . 1 . 1 0 , 6 equiv of di-2-pyridyl carbonate (DPC), and 2 equiv of 4(dimethylamino)pyridine (DMAP) at 80 ~ for 100 h produced the C-2', C-7protected 2.1.12 in 80% yield (yield based on only 50% conversion). The protecting groups were removed by using 0.5% HC1 in ethanol to give Taxol| in good yield (Scheme 3). This method suffers from two major drawbacks: esterification required excess amounts of the expensive chiral amino acid (6 equiv or more) and only 50% conversion was observed even under forcing conditions [10].

[Scheme 311 OAc

O

_

Ph

HO ....

NH --

O

i

ii

p hv- ~ _, , ~ O H

.o" BzO

~c

~

o.yo

2.1.11

2.1.10

O P h ' ~ NH

O

3 ii

P h ~ O

....

,

Taxol, 1.1.1

~ 0

2.1.12 Conditions: (i) DPC, DMAP, PhMe, 80 ~

80%; (ii) HC1, EtOH/H20, 0 ~

89%.

The esterification step was significantly improved by Commer~on and co-workers. The phenylisoserine side chain was introduced as an oxazolidine,

260 which

underwent

esterification

under

standard

DCC/DMAP

coupling

conditions in high yield [22]. The methodology avoided the use of an excess of enantiomerically pure amino acid 1.1.2, and the coupling yield was over 90%. The key intermediate was again a chiral epoxide (2.1.15, a homolog of 2.1.2), which was synthesized by condensation of the boron enolate of (4S, 5R)-3b r o m o a c e t y l - 4 - m e t h y l - 5 - p h e n y l - 2 - o x a z o l i d i n o n e (2.1.13) w i t h b e n z a l d e h y d e followed by t r e a t m e n t with lithium ethoxide, to produce chiral epoxide 2.1.15 in high optical purity.

Scheme 411

0 0 BrV~NA O \ / ,, ~ -~ Ph

OH 0 0 i = p h ~ ~ J ] ~ N~]O Br ~ ,~ -" Ph

2.1.13

2.1.14

N3 0 P h / ~ ~ - OEt

ii

0 r_ ph~--~CO2Et 2.1.15

Boc,.

NH2 .0. iv

= p h , / ~ ~_

OEt

iii

v

NH 0

P h Z ~ ~ - OEt

vi

_

OH

2.1.16

OH

OH

2.1.17

2.1.18

Ph~.~,- "c02H

Ph~}~.~,,-"C02Et vii

Boc~ N ~ O 2.1.19

Boc~N~ 0 2.1.20

Conditions: (i) NEt3, Bu2BOTf, CH2C12,-70 ~ to rt, then PhCHO,-78 ~ to 0 ~ 58%; (ii) EtOLi, THF, -75 ~ to 15 ~ 81%; (iii) NAN3, EtOH, NH4C1, 60 ~ 99%; (iv) H2, Pd/C, EtOAc, 86%; (v) Boc20, NaHCO3, CH2C12, 20 ~ 76%; (vi) CH2=C(Me)OMe, PTSP, PhMe, 80 ~ 99%; (vii) LiOH, EtOH/H20, 20 ~ 100%.

Epoxide 2.1.15 was converted to N-protected amino ester 2.1.18 via the azide method. Conversion to acetonide 2.1.19 was followed by saponification to afford

261 the free acid 2.1.20 in high yield (Scheme 4). T r e a t m e n t of 1.5 equiv of 2.1.20 with 2.1.22, using 1.6 equiv of DCC and 0.5 equiv of DMAP in toluene at 80 ~ afforded high yields of 2 . 1 . 2 4

without

any

detectable

epimerization.

Deprotection using formic acid followed by N-acylation and removal of the Troc group afforded Taxol | in high yield. Taxotere | (2.1.29) was similarly prepared (Scheme 5).

[ Scheme 5 OR~

0

O =

HO.... H d B ~

i:I(~A'~cO

Ph~ i

O"'

Boc-~_O

HO BzO 2.1.23 RI=CO2CH2CCI 3 2 . 1 . 2 4 R1 = Ac

Troc = C02CH2CCI 3

OR 1 O N_H2 0 ph/'Z',.,v~ O.... -

~

OR1 0 0 T r o c

.... J]~

I

2.1.21 RI= CO2CH2CCI 3 2.1.22 R 1 = Ac

ii

,

II

iii

~

~ S

o.

H

121

O

2.1.25 RI= CO2CH2CCI 3 2 . 1 . 2 6 R] = Ac FI10 R2HN Ph

O _ OH

R2HN

OH o

0 .... OH H d ~ ~BzO .:

2.1.27

O

RI=CO2CH2CC]3, 2.1.28 RI=AC , R2=Bz

I~I(~A~cO

R2=Boc

O

....

- ,

O BzO OAc 2.1.29 RI=H, R2=Boc (Taxotere| 1.1 1 RI=AC, R2=Bz (Taxo]|

(i) DCC, DMAP, PhMe, 2.1.20, 80 ~ 98%; (ii) HCO2H, 20 ~ 78% (2.1.25), 80% (2.1.26); (iii) Boc20, NaHCO3, THF, 20 ~ 87% or BzC1, NaHCO3, EtOAc, 87%; (iv) Zn, AcOH, MeOH, 60 ~ 89-90%. Conditions:

262 In a following paper, Greene has shown t h a t even the C-2 epimer of 2.1.20 can function as a suitable precursor to Taxol | and Taxotere | Indeed, C-2 epimerization during the acylation reaction is virtually complete, and this opens the possibility of using, as esterifying agents, side chain precursors that are not stereochemically homogeneous at C-2 [23]. In a similar approach, Didier and co-workers prepared diastereomeric mixtures of oxazolidinecarboxylic acids 2.1.30 by treating amino ester 2.1.9 with chloral [24]. Coupling of 2.1.30 with 2.1.21 by the DCC/DMAP procedure produced 2.1.31. Treatment with zinc in acetic acid removed the two carbonate groups and hydrolyzed the oxazolidine to provide 2.1.32, which was converted to Taxotere | by the usual procedure (Scheme 6).

[Scheme 6~ CCl 3

I

HN~"O

Ph"

i =

CO2H

2.1.30 NH 2 .O

I

0130~ ~ , , , _ . O ' I~ Hn H

OH

TrocO~,

O i1|

HO Bz()

2.1.31

O

\

/O OTroc

OH iii

2.1.29

OH HO

-

BzO

"

~Ac

2.1.32 Conditions: (i) 2.1.21, DCC, DMAP, PhMe, 99%; (ii) Zn, AcOH, EtOAc, rt; (iii) Boc20, MeOH, rt, 70% overall.

Jacobsen's approach to the Taxol | side chain employed (salen)Mn(III) complex 2 . 1 . 3 3 to effect the asymmetric epoxidation step [25]. Partial h y d r o g e n a t i o n of commercial ethyl phenylpropiolate (2.1.34) to Z - e t h y l c i n n a m a t e (2.1.35) using Lindlar's catalyst followed by epoxidation with commercial bleach in conjunction with 2.1.33 afforded the Z - ( R , R ) e p o x i d e 2.1.15 in >95% e.e. The trans isomer of 2.1.15, however, was a significant byproduct in the epoxidation reaction. Nonetheless, in a highly regioselective

263 ring-opening process, the mixture of cis and trans epoxides gave 2.1.36 in high yield upon t r e a t m e n t with ammonia. Intermediate 2.1.36 was subsequently transformed into the desired product 1.1.2 under standard conditions. The low cost of the reagents makes this an attractive and practical approach to the side chain (Scheme 7).

Scheme 7 / O'McI%

tBu

tBu

U

2.1.33 0 Ph

~

CO2Et

i =

2.1.34

Ph\

/CO2Et

ii

~

h~--~C P O2Et

2.1.35

2.1.15 O

NH 2 Q iii=

Ph

: OH _

2.1.36

NH 20. NH 2 ~

,v

Ph

: OH

2.1.37

P h / [ L NH OH

v

=

O

ph/~.~_

OH

OH

1.1.2

Conditions: (i) H2, Lindlar cat., 84%; (ii) NaOC1, (R,R)-2.1.33 (6 tool %), 4-phenylpyridine-Noxide (0.25 equiv), CH2C12, 56%, >95% e.e.; (iii) NH3, EtOH, 100 ~ 65%; (iv) Ba(OH)2, then H2SO4, 92%; (v) PhCOC1, NaHCO3, then HC1, 74%.

Sharpless reported a six-step asymmetric synthesis of the Taxol| side chain using the novel chiral catalyst (DHQ)2-PHAL [26]. Asymmetric dihydroxylation of 2.1.6 afforded diol 2.1.38 in 99% e.e. In a one-pot procedure, 2.1.38 was protected as a cyclic orthoester and regioselectively opened by acetyl bromide to produce a 6:1 mixture of bromo acetates, favoring the desired stereoisomer 2.1.39. T r e a t m e n t of 2.1.39 with sodium azide followed by hydrogenation gave the N-acetyl derivative 2.1.40 which was converted to 1.1.2 in fair overall yield (Scheme 8).

264 A very similar approach was also reported by Koskinen, although the enantioselectivity of the dihydroxylation step was not reported [27]. Utilization of Z-cinnamates in this approach would eliminate the need for the double inversion operation at C-3, but these substrates are dihydroxylated in very poor e.e. [27].

[ Scheme 8/ 0 ph

v

OH OCH3

0

Br

OH

2.1.6

CH 3

2.1.39 0

Me'~NH

iii

OCH 3 _ OAc

~

2.1.38 0

0

0

p h ~ l J L.. O C H OH

2.1.40

Ph'~NH

3

iv, v

0

ph..~.jl ~ OH

(~H 1.1.2

Conditions: (DHQ)2-PHAL (0.5 tool %), K2OsO2(OH)4, NMMO, t-BuOH, rt, 72% (99% e.e.); (ii) MeC(OMe)3,p-TsOH, CH2C12, rt, then AcBr, CH2C12,-15 ~ 60%; (iii) NAN3, DMF, 50 ~ H2, Pd/C, MeOH, rt, 74%; (iv) 10% aq. HC1, heat; (v) PhCOC1, 2 N aq. NaOH, rt, 72% overall. Potier and his group attempted an oxyamination reaction directly on 13cinnamoyl baccatin 2.1.41 in order to develop a semi-synthesis of Taxol | and Taxotere (ii) [28]. Negligible stereoselectivity was observed when employing the classical Sharpless catalytic procedure; two pairs of threo and erythro isomers 2 . 1 . 4 2 - 2 . 1 . 4 5 were obtained in poor yields along with recovered starting material (ca. 50%). The isomers were separated and converted to Taxol | and Taxotere | derivatives using standard conditions. This procedure allowed the synthesis of all possible diastereomers and regioisomers for SAR studies (vide infra) (Scheme 9). 6.2.2. Chiral Pool Approach Greene and collaborators employed (S)-phenylglycine (2.2.1), available from the natural chiral pool, as a starting material in the synthesis of Taxol | and Taxotere (i!) side chains [29]. Aminoacid 2.2.1 was reduced to the alcohol,

265 followed by in s i t u protection of the amino group to afford 2.2.2 or 2.2.3. An a t t e m p t at a t a n d e m S w e r n oxidation/addition of v i n y l m a g n e s i u m bromide to the crude aldehyde g e n e r a t e d racemic 2.2.5.

Scheme 9/ AcO ~r~

O Ph

o,k_

O ~~

r~

s .....~ ~ . ~ k

AcO

R2 .O

;

__~ Ph

O

OTroc

o ....

"' Ho" o~z~ ~ ~ o HO OBz 2.1.41 2.1.42 (2'R,3'S)R]=OH, I~2=NHCO2tBu 2.1.43 (2'S,3'R)RI=OH, I~2=NHCO2tBu 2.1.44 (2'R,3'B)RI=NHCO2tBu,R2=OH 2.1.45 (2'S,3'R)RI=NHCO2tBu, R2=OH =

Conditions: (i) t-BuOCONCINa, AgNO3, OsO4, H20/CH3CN.

[Scheme 1011

0

N_H2

RJJ~NH i =- ~ O H

=

~ ~ O

0

OH

RflL'NH ii =

H

'L2

2.2.1 O

2.2.2 R = Ph 2.2.3 R = Ot-Bu

R~ N H iii

R~ N H iv, %

2.2.5 R - P h 2 . 2 . 6 R = Ot-Bu

2.2.4 O

-

0 -

OH

2.1.10 R = Ph ~

2.2.7' R = Ot-Bu

Conditions: (i) LiA1H4, PhCOC1 or Boc20, 74-79%; (ii) (COC1)2, DMSO, i-Pr2NEt, CH2C12,-78

~ to-35 ~ (iii) CH2=CHMgBr, -78 ~ RuC13, NaIO4, NaHCO3, 62-82%.

62% overall; (iv) CH2=CHOEt, H3 O+, 90%; (v) cat.

266 However, by adding the Swern oxidation product to the vinylmagnesium bromide in a m i x t u r e of T H F - C H 2 C 1 2 gave 2 . 2 . 5 with good s y n diastereoselectivity (9:1) and no racemization. Intermediate 2.2.5 was further elaborated to 2.1.10. Side chain 2.2.7 was similarly synthesized (Scheme 10). 6.2.3. Lithiobenzylamine Method A conceptually novel approach to the synthesis of the Taxotere | side chain employed the dianion of N-Boc-benzylamine 2.3.1 [30]. Treatment of such dianion with acrolein produced a 6:1 mixture of amino alcohols with a bias for the desired s y n alcohol 2.3.2 . The diastereomers were separated and the s y n alcohol was protected as a (trichloroethoxy)methyl ether, 2.3.3. Oxidative cleavage of 2.3.3 gave a racemic acid which, upon resolution using (+) ephedrine, gave the optically pure enantiomer 2.3.4 (Scheme 11). Scheme 11/

NHBoc

Boc.

Boc. NH -

i =

-

ii

~

NH_

. ~ O w C C i

2.3.1

2.3.2

3

2.3.3

Boc.NH 0 II1,1v ....

_

-

~

OH 0 ~ CCI3

2.3.4

(i) sec-BuLi, acrolein, THFfrMEDA,-78 ~ 49%; (ii) BrCH2OCH2CC13, proton sponge, CH3CN, 70 ~ (iii) RuC13, NaIO4, NaHCO3, CC14/ CH3CN/H20, rt, 80%; (iv) (+)ephedrine, 83%. Conditions:

6.2.4. Diastereoselective Michael Approach Davies reported a synthesis of the Taxol|

side chain

based upon

asymmetric tandem conjugate addition-electrophilic hydroxylation of

tert

butyl

cinnamate 2.4.1 [31]. The chemistry involved conjugate addition of the homochiral lithium (S)-(a-methylbenzyl)-benzylamide 2.4.2 to 2.4.1, followed by hydroxylation

of the i n t e r m e d i a t e

enolate

with

(+)-(camphorsulfonyl)

267 oxaziridine to produce the a n t i product 2.4.3 in high diastereoselectivity. In order to adjust the stereochemistry at C-2, the intermediate 2.4.3 was then subjected to hydrogenolysis followed by methanolysis and benzoylation, to afford the a n t i hydroxy amide 2.4.4 in high yield. Multi-step inversion at C-2 via oxazoline 2.4.5 gave the well-known methyl ester 2.1.5 in high yield and optical purity (Scheme 12). [Scheme 1211

-"

0

Ph

Ph

"

OBut

//~" N J

+ ph/~N/~Ph Li

2.4.1

/

i ~ Ph

OBut 2.4.3

Ph

Ph'~NH

O

Ph

O

N,~O OMe

OH

ii, iii, iv

OH

2.4.2

O

0

Ph'~NH

=-

OMe Ph

-

Ph

O : OH

OMe

O 2.4.4

2.4.5

2.1.5

Conditions: (i) (+)-(Camphorsulfonyl)oxaziridine, THF,-78 ~ 89%, >98% d.e.; (ii) H2, Pd/C, AcOH; (iii) HC1, MeOH; (iv) PhCOC1, NEt3, 96% overall; (v) DEAD, PPh3, THF, 0 ~ 77%; (vi) HC1, MeOH; (vii) NaHCO3, 91% overall.

6.2.5. The ~-Lactam Method The racemic synthesis of the Taxol |

side chain v i a ~-lactams was

reported by Palomo and co-workers; their chemistry utilized as a key intermediate azetidine-2,3-dione 2.5.1 [32], which in turn can be prepared by a number of [2+2] protocols [32, 33]. Diastereoselective reduction of 2.5.1 using sodium borohydride afforded exclusively cis lactam 2.5.2. Protection of the C-3 hydroxyl group followed by oxidative removal of the N-aryl group furnished 2.5.3. Ring opening of the ~-lactam with chlorotrimethylsilane in methanol afforded the key intermediate a-hydroxy-~-amino ester 2.5.4, which was then converted to 2.1.5 in the usual way (Scheme 13) [32]. The first direct application of ~-lactams in the semi-synthesis of Taxol| was demonstrated by Holton [34]. Treatment of 2.1.11 with n-BuLi at-40 ~ in

268 THF chemoselectively generates the alkoxy anion at the C-13 position, and this readily attacks r a c e m i c [~-lactam 2 . 5 . 5 to afford a m i x t u r e of chromatographically separable diastereomers 2.5.6 and 2.5.7 in high yield.

[Scheme 13 ]1 O

O

Ph

H O,,,, i

,,,Ph I N

,,

oJ

C I"'~O,,,, II, II1=

.....

OMe 2.5.2

.~

NH 2 .C) =

2.5.3

OMe

2.5.1

iv

Ph

~

oJ

: OH

Ph

v

O Me

,,,Ph I N.

=

NH Ph

U

OH

2.5.4

O Me

2.1.5

Conditions:(i) NaBH4, THF/MeOH; (ii) CICH2COC1, Py, CH2C12, 80% overall; (iii)

(NH4)2Ce(NO3)6, CH3CN/H20, 0-5 ~ -70 ~ to 20 ~ no yield.

70%; (iv) TMSC1, MeOH; (v) PhCOC1, NEt3, CH2C12,

Scheme 14/ OAc Et3SiO,"

tS

.:.

,Ph

i

0

Ph

HO BzO 2.1.11

(+)2.5.5

(~SiEt3

OSiEt3

+ HO ....

oJ-'

BzNH O .-= II phJ"-.,v.--'~-_ O ....

O

OAct 2 0 S i E t 3

OAc BzNH

,%

+ O

2.5.6 Conditions: n-BuLi or LiHMDS, -40 ~ to 0 ~

O

O

ph-~~O

t3 ....

OS,E, 2.5.7 80-90% of 2.5.6+2.5.7.

H

~

0

269 Furthermore, when excess racemic 2.5.5 (ca. 5-7 equiv) was used, an 8:2 mixture of diastereomers, with a bias for the desired 2'R, 3'S diastereomer, was isolated (Scheme 14). However, the use of excess ~-lactam and the c h r o m a t o g r a p h i c separation of the diastereomers can be avoided if optically pure cis (3R, 4S)~lactam is employed. Thus, t r e a t m e n t of 2.1.11 with 1.6-1.8 equiv of resolved, enantiomerically pure 2.5.5 in the presence of n-BuLi afforded only 2.5.6 in high yield [34]. Holton's semi-synthetic approach to Taxol| therefore, required a practical synthesis of enantiomerically pure ~-lactams. This was reported by Ojima, Georg and co-workers [35-37]. Their c h e m i s t r y relied on an enantioselective enolate-imine cyclocondensation to synthesize the required 3hydroxy-4-aryl-~-lactams. T r e a t m e n t of the enolate of ester 2.5.8, bearing Whitesell's chiral auxiliary [(-)trans-2-phenyl-l-cyclohexanol)] with silylimine 2.5.9 gave exclusively 2 . 5 . 1 0 in high enantiomeric purity (96-98% e.e.). Conversion to 1.1.2 was accomplished under standard conditions (Scheme 15).

[Scheme 1511

Ph

0 /~OTIPS

Ph +

I(

TIPSO, "

i

Me3si/ 2.5.8

2.5.9

0 iv

Ih111 =

NH2~

ph~ V OH .

OH

2.1.37

-HCI

o,7

"H

2.5.10

ph ) 1 " NH

=

Ph "

0

ph/~~]-OH OH

1.1.2

Conditions: LDA (2 equiv), THF, -78 ~ to rt, 85% (96% e.e.); (ii) Bu4NF, THF, rt, 98%; (iii) 6 N HC1, reflux, 100 ~ (iv) PhCOC1, aq. NaHCO3, CH2C12, 70%. (TIPS=triisopropylsilyl). This versatile approach was also used to synthesize a number of taxane analogs modified at the side chain (vide infra). In a modification of the above protocol, Oppolzer's (-)-10-dicyclohexyl sulfamoyl-D-isoborneol was used by

270 Georg and collaborators to prepare enantiomerically pure 2.5.13, the Holton intermediate [38].

High enantioselectivity was observed in this case also

(Scheme 16). Georg and co-workers reported the first attempt at using an asymmetric Staudinger reaction to prepare the required optically pure 13lactam.

Using

a peracylated galactopyranosyl template, these workers

initially reported that a single diastereomer, 2.5.16, was obtained [39]. A later study, however, demonstrated that this chiral template affords little or no diastereoselectivity in the Staudinger reaction (Scheme 17) [40].

[Scheme 1611

Ph O SO2N(C6H11)2

2.5.11

TBSO,

Ph

NTMS

TBSQ

N- H O

2.5.9

2.5.12

,Ph N Ph "~ O

2.5.13

Conditions: (i) LDA, THF, 94% (97% e.e.); (ii) PhCOC1, NEt3, DMAP, CH2C12, 96% (TBS= t-

Butyl dime thylsilyl).

[Scheme 1711 OAc ,...-OAc Ph

AcO

OAc

H

ArO~ 0

2.5.14

CI

2.5.15

OAc ,~OAc O AcO

OAc ~.~OAc O Ar

Ph

2.5.16

i

AcO

_ Ph

2.5.17

Conditions: NEt3, CH2C12, rt, 75% (2.5.16:2.5.17= 2:3) [Ar=p-methoxyphenyl].

Ar

271 A highly diastereoselective approach to the Taxol | side chain via the Staudinger reaction was reported by Farina and co-workers [41]. The reaction between L-threonine-derived imine 2.5.18 and the acid chloride 2.5.19 under typical Staudinger conditions afforded the desired cis ~-lactam 2.4.20 in high yield and with good diastereoselection ( 3 R , 4 S / 3 S , 4 R >10:1). The chiral template was removed by t r e a t m e n t of 2.5.20 with t e t r a b u t y l a m m o n i u m fluoride, followed by mesylation/elimination to give 2.5.22 quantitatively; this was then ozonized to 2.5.23, and finally base-promoted hydrolysis of the acetate and the oxalamide groups afforded the target compound 2.5.24 in high optical purity (99.5%). Lactam 2.5.24 was eventually used in the preparation of Taxol | using Holton's protocol (Scheme 18).

{Scheme 18 ]J

o= =

,co

N..~~

+

i

=

,COoj"" -

O-" -Cl

CO2Me 2.5.18

,COoj"" -

OR ii N~~,,,. "I

"

CO2Me 2.5.19

2.5.20

"

I

OH :

N~-.~ CO2Me 2.5.21

R=SiPh2OBu t

m

,...

AcO.,,,

,,,Ph

iii

AcO,%

,,,Ph

N.O COeMe

2.5.22

CO2Me

2.5.23

HO,,,,

,,,Ph N. H

2.5.24

Conditions: (i) NEt3, CH2C12,-40 ~ to rt, 74%; (ii) Bu4NF, THF, AcOH, 82.5%; (iii) MsC1, NEt3, CH2C12, -78 ~ to rt; (iv) 03, CH2C12, -78 ~ the Me2S; (v) aq. NaHCO3, MeOH, 80% overall.

Bourzat and Commerqon reported a moderately diasteoselective Staudinger reaction using (S)-a-methylbenzylamine as chiral template [42]. The chiral imine 2.5.25 was treated with acetoxyacetyl chloride 2.5.19 in the presence of triethylamine, to give a 3:1 mixture of 2.5.26 and 2.5.27. This

272 mixture

was

subjected

to base-catalyzed hydrolysis

and

the

desired

diastereomer 2.5.28 was isolated by crystallization. Hydrolytic cleavage followed by removal of the chiral auxiliary by hydrogenation afforded the ester 2.1.9 (Scheme 19). This approach could also be used to prepare a number of analogs to be used for SAR purposes (vide infra).

(,Scheme 19]l AcQ i

Ph..11 Nv

""

,Ph " Nv Ph :

0~.._ I

Ph OH 3

0

+

Ph

~

Ph

Ph _ OH 3

2.5.27

H3C~NH

OH3

2.5.28

ii

0,/~- Nv

2.5.26

,Ph Nv

Ph

CH 3

2.5.25 HO,,,,

AcO.

0 : OH

B~ 0 Me

-

2.5.29

Ph

0 : OH

0 Me

2.1.9

Conditions: (i) AcOCH2COC1, NEt3, CHC13, 0 ~ to rt, 74% of 2.5.26 + 2.5.2"/(3:1); (ii) 1 M aq. KOH, THF, 0 ~ 67%; (iii) 6 N HC1, MeOH, reflux, 88%; (iv) H2, Pd/C, MeOH/AcOH then Boc20, CH2C12,aq. NaHCO3, 20 ~ 70%. Holton's asymmetric Staudinger approach to the synthesis of chiral cis3-hydroxy-4-arylazetidinones utilized the Evans strategy [43]. Under the Staudinger conditions, chiral acid chloride 2.5.30 and imine 2.5.31 afforded 2.5.32 in high yield and complete diastereoselectivity. Treatment of 2.5.32 with LiHMDS in dichloromethane followed by the addition of N-chlorosuccinimide gave 2.5.33 as a mixture of diastereomers, which were eventually converted into 2.5.1. Diastereoselective reduction using sodium borohydride as previously described produced the chiral alcohol 2.5.2. Hydroxyl group protection, Ndearylation and benzoylation afforded 2.5.36 (Scheme 20). A short synthesis, amenable to large scale production, of racemic 4-aryl and 4-heteroarylazetidin-2-ones was reported by Rey and coworkers at BristolMyers Squibb. Treatment of commercially available hydrobenzamide 2.5.37

273 with acetoxyacetyl chloride in the presence of triethylamine afforded cis ~lactam 2.5.38 as a diastereomeric mixture (at the aminal carbon) in good yield. Removal of the N-benzyl group by hydrogenation afforded racemic 2.5.39 (Scheme 21) [44].

[Scheme 20]1

pPh

0

c,

I~ Ph

/'~ O. N,,

+

Ph ,,,

O oJ_,

i.__

MeO

2~~. "

2.5.311

2.5.31

pPh

2.5.3

OMe

pPh

.

o

O.~N,,,

i___Li... " ~

2.5.33

OMe

HO% ,,,Ph N

vi, vii

2.5.2 ~

I

iv

OMe

2.5.34

EEO,,,, 0# 1

0 0~"

,,, N.

2.5.35

Ph

,Ph v

2.5.1 EEO,%

r

viii

H

OMe

0

,,"

Ph

N.

COPh

2.5.36

OMe Conditions: (i) NEt3, CH2C12, -78 ~ 93%; (ii) LiHMDS, CH2C12, -78 ~ NCS, 95%; (iii) aq. AgNO3, CH3CN, 0 ~ (iv) SiO2, >95% overall; (v) NaBH4, MeOH, 0 ~ 100%; (vi) Ethyl vinyl ether, MsOH, 0 ~ 100%; (vii) (NH4)2Ce(N03)6, CH3CN, 87%; (viii) PhCOBr, Py, CH2C12, 0 ~ 98%. [EE=l-Ethoxyethyl].

6.2.6. Chiral Sulfinimine Approach Davis and co-workers described the use of chiral sulfinimines in their enantioselective approach to the Taxol | side chain [45]. Addition of the lithium enolate of methyl acetate to readily available, enantiomerically pure

274 2.6.1, followed by desulfinylation/benzoylation, afforded N-benzoyl-~-amino ester 2.6.3 in good yield.

[Scheme 21 ]~ Ph"71

Ir

/ Ph

NT~N Ph

A c (3, , Ph "" " 0,/~,._ NI ~ Ph y

i

A cQ ii

r

2.5.37

""

""

Ph

0/I/_ NI

=

H

2.5.38 N 7

2.5.39

Ph Conditions: (i) AcOCH2COC1, NEt3, EtOAc, 5 ~ >95%; (ii) H2, Pd/C, EtOAc, 78%.

S u b s e q u e n t a s y m m e t r i c h y d r o x y l a t i o n u s i n g (+)-(camphorylsulfonyl) oxaziridine gave a 86:14 s y n : a n t i mixture of 2.1.5 and 2.4.4 in fair yield. Chromatographic separation of the diastereomers afforded 2.1.5 in good enantiomeric purity (>93%) (Scheme 22).

[Scheme 2211

O

O

I

O

H

Ph~ "N

ph~S'NH Ph

Ph

2.6.1

0

OMe

2.6.2

A N__~~

i v _ Ph "-

Ph

ph.fl

O

. OMe OH 2.1.5

ph - - ~ ~ O M e 2.6.3

0

AN

+ Ph

~

Ph

OMe OH 2.4.4

Conditions: (i)CH2=C(OLi)OMe, -78 ~ 76%; (ii) CF3CO2H, MeOH; (iii) NEt3, DMAP, PhCOC1, 76% overall; (iv) LDA, LiC1, (+)-(camphorylsulfonyl)oxaziridine, -100 ~ to -78 ~ 58%.

6.2.7. Aldol Reaction Approaches A n u m b e r of asymmetric aldol-type approaches have been used to prepare taxane side chains. Three basic strategies have been utilized: a) Use of

275 a chiral aldehyde or imine (or equivalent) in conjunction with an achiral enolate; b) Use of a chiral enolate equivalent in conjunction with an achiral aldehyde or imine; c) Use of a chiral catalyst to promote reaction between achiral partners.

Combinations of the above three strategies are also

conceivable. Hanaoka and his group employed optically pure (+)-tricarbonyl(q6-2trimethylsilylbenzaldehyde)chromium (0) complex 2.7.1 in conjunction with the titanium enolate of thioester 2.7.2

to afford, after desilylation and

decomplexation, the a n t i - a l d o l product 2.7.4 in a highly diastereoselective manner. Mitsunobu reaction with hydrazoic acid gave the desired s y n azide 2.7.5 in high yield. Reduction with triphenylphosphine and water furnished the amino derivative which was benzoylated to afford 2.7.6 in high optical purity (> 98%). Deprotection with thallium nitrate in methanol followed by hydrogenolysis gave the target compound 2.1.5 (Scheme 23) [46,47]. [Scheme 2311

OH

0

o + Cr

TMS

oc'lco

_

OBn

2.7.1

CO

;

SBu t

Cr

Bu t

..... //,i//

TMS

oc'l'co

2.7.2

s

2.7.3

CO

O

.JL OH

O

N3

/~~,._

P h ~ S B u OBn 2.7.4

iv

t

=

Ph~

Ph

:

Ph--NH SB

OBn 2.7.5

0 Ph'~NH

vii

~

O

_

OBn

2.7.7

v, vi~

Ph~

Ph/[L'NH OMe

viii

=

Ph

:

SBu t

OBn 2.7.6

0

O

~v ~

Ut

O

O _

OMe

OH

2.1.5

Conditions: (i) TiC14,NEt3, CH2C12, -78~ 93%; (ii) Bu4NF, HF, CH3CN/THF, -78~ to 0~

(iii) hv, Et20,0~ 63% overall; (iv) HN3, PPh3, DEAD, C6H6, rt; (v) PPh3, H2OfrHF, 60~ (vi) PhCOC1, DMAP, CH2C12, 0~ 53% overall; (vii) Tl(NO3)3, MeOH, rt, 90%; (viii) Pd/C, H2, EtOH, 60~ 89% (>98% e.e.)

276 Yamamoto's approach to the synthesis of 2.1.5 employed a double diastereoselection strategy utilizing chiral Lewis acid 2.7.9 [48]. Reaction of chiral imine 2.5.25 with a-silyloxy (Z)-ketene acetal 2.7.8 mediated by chiral boron reagent 2.7.9 produced enantiomerically pure syn adduct 2.7.10 (syn/anti = 99/1, diastereofacial ratio = 99/1). Hydrogenolysis followed by benzoylation under Schotten-Baumann conditions gave N-benzoyl-(2R,3S)-phenylisoserine methyl ester (2.1.5) in good yield (Scheme 24).

[Scheme 24]l Ph ~

N~

Ph OMe .--/~-~0 + (Et)3SiO Si(Et) 3

Ph

Me

~NH i -_ (S)-2.7.9

2.7.8

2.5.25

0

phi2-< k_ OMe OH 2.7.10

0 il, i l i

P h / ~ NH

0

w -

.

ph~~l"OMe OH

2.1.5 Conditions: (i) CH2C12,-78 ~

~~]"-0

(S)-2.7.9

91%; (ii) H2, Pd/C, MeOH; (iii) PhCOC1, aq. NaOH, THF, 68%

overall.

Approaches utilizing chiral enolates were reported by Swindell and Greene. Swindell and coworkers attempted an asymmetric hetero-Diels-Alder reaction between N-benzoylbenzaldimine 2.7.11 and chirally modified ketene acetal 2.7.12 [49]. Aqueous work-up furnished, however, aldol products as a mixture of diastereoisomers, with 2.7.13 as the major product. The isomers were subjected to debenzylation followed by transesterification to provide a mixture containing mainly 2.1.5 (93:7 syn/anti, only one syn isomer) (Scheme 25). One should note that several chiral alcohols were examined as templates for the above operation and (1S,2R)-(+)-trans-2-(1-methyl-l-phenylethyl)-lcyclohexanol was the best one. Greene's approach utilized the Oppolzer template. Camphorsultam 2.7.14 was condensed with benzaldehyde N-(t-

277 butoxycarbonyl)imine 2.7.15, to provide exclusively 2.7.17 with complete (>99%) enantio- and diastereoselection. The chiral auxiliary was then oxidatively cleaved, to afford 2.7.17 in fair overall yield [50] (Scheme 26).

[Scheme 25]l

O

Me Ph.,,~ Me (Me)3SiO~L_ / ~ + BnO/_~ 0

O Ph~N//'-.ph 2.7.11

Ph/~NH i

Me O Ph.J/Me OBn

2.7.13

2.7.12

0 Ihtll

Ph'~ NH 0 Ph~OMe 2.1.5 OH

Conditions:

(i) C6H6, rt, 75%; (ii) Pd(OH)2, H2; (iii) MeONa/MeOH, 82% overall.

{scheme 26]l

O

BocHN O N

+

=

OBn

NBoc

02 2.7.14 BocHN

2.7.15

Ph

_ N OBn 02 2.7.16

O

Phi_

OH OBn

2.7.17 Conditions:

(i) LiHMDS, THF,-78 ~ 66%; (ii) LiOH, H202, rt, then aq. Na2SO3, 0 ~ 70%.

6.2.8. Enzymatic Approaches The s y n t h e s i s of all d i a s t e r e o m e r s of 3 - p h e n y l s e r i n e s and 3phenylisoserines in enantiomerically pure form using enzymatic resolution was first reported by HSnig [51]. Racemic butyryl ester 2.8.3, synthesized as

278 shown below in Scheme 27, was resolved by hydrolysis with P s e u d o m o n a s fluorescens. Both the alcohol 2.8.4 and the unreacted isomer 2.8.5 were isolated in high enantiomeric purity (>98% e.e.). Sih and co-workers reported the enzymatic resolution of 3-acetoxy-4phenyl ~-lactams 2.8.6-2.8.8 using bacterial lipases [52]. The most suitable lipase for the various transformations was the P s e u d o m o n a s lipase P-30. Under these conditions, the undesired enantiomer was selectively hydrolyzed.

[Scheme 27]~

0

N3 i, ii

ph~~-'~OE t

0

N3

= Ph"'Z"'~OEt==

+

0

Ph~OEt

2.8.1

N3

0

OH

2.8.4

N3

0

O\ ~

2.8.5

0

Conditions: (i) NAN3, aq. EtOH, NH4C1, 60%; (ii) (n-PrCO)20, py, H20, 92%; (iii) Pseudomonas fluorescens, 2.8.4 (26%, >98% e.e.), 2.8.5 (35%, >98% e.e.). These intermediates were converted to the C-13 Taxol | side chain 1.1.2 by standard protocols (Scheme 28). Patel and co-workers at Bristol-Myers Squibb have optimized this resolution on a large scale and have applied it to the commercial production of Taxol| [53]. Chen has enzymatically resolved racemic trans-phenylglycidic ester 2.8.9 by transesterification with lipases in organic media. Thus, incubation of racemic 2.8.9 with Lipase MAP-10 in hexane-isobutyl alcohol (1:1) afforded 2.8.10 and 2.8.11 in high enantiomeric excess. The product and the substrate were separated by fractional distillation. The individual enantiomers were subsequently converted to the Taxol| side chain 2.1.5 in a number of steps. It is noteworthy that both 2.8.10 and 2.8.11 were converted to optically pure 2.1.5, as shown in Scheme 29 [54].

279 Chen's chiral oxazoline 2.4.5 was utilised by Kingston in a new semisynthesis of Taxol| saponification of 2.4.5 with aqueous NaOH provided 2.8.14, which upon treatment with 7-TES baccatin 2.1.11 underwent a smooth coupling reaction to afford 2.8.15. Acid-catalyzed hydrolysis afforded Taxol| in good yield [55] (Scheme 30).

[ Scheme

2811

II

Ac O,,,, ,,,Ph

AcO,,,,

N

.,"Ph

oJ-' N OCH3 Ph

,,"Ph N

(+)-2.8.7

""~"~

ACE),,,,

O

Ph (+)-2.8.8

N ~ Ph

OH OH .

1.1.2 A Bristol-Myers Squibb group reported the synthesis of chiral phenylisoserine ethyl ester 2.8.17 via diastereoselective microbial reduction of the prochiral ketone 2.8.16 [56]. Microorganisms from H. polymorpha SC 13865 and H. fabianii SC13894 effectively reduced the ketone in high yield (>80%) and optical purity (>95%) (Scheme 31). Similarly, Kayser and Kearns used a yeast-mediated stereospecific reduction of chiral (~-keto ester 2.8.20, obtained in three steps from natural (S)phenylglycine 2.2.1, as shown in Scheme 32. Under these conditions, the desired 2.5.4 was obtained diastereomerically pure, although no information on the optical purity of this intermediate was reported [57]. Standard transformations were used to convert 2.5.4 into side-chain synthon 2.1.5, although no details of the experimental procedures used are given.

280

6.3. S I D E

CHAIN

MODIFICATIONS

FOR

STRUCTURE-ACTIVITY

RELATIONSHIP STUDIES 6.3.1. Simplified Side Chain Analogs Studies

of n a t u r a l

and

semisynthetic

congeners

of Taxol |

have

d e m o n s t r a t e d t h a t a taxane ring and an ester C-13 side chain are required for a n t i t u m o r activity, since b a c c a t i n

III 1.1.3 and N - b e n z o y l

(2'R, 3'S)-3'-

phenylisoserine 1.1.2 are devoid of significant activity [58]. [ S c h e m e 29~

ph\O \

i \

0 =

CO2Me

0

P h " ' L ' ~ c O2Me +

2.8.9

PhJ/---&"CO2Bu i

2.8.10

N3 ii, iii 2.8.10

2.8.11

O

BzHN

~

=-- Ph

:

OMe

=

Ph

_

OH N3

v//i,/x

ph./~CO2Bu, OH

O

2.8.12

./~ Ph NH

N3 vii__

ph/~~CO2Bui OCOPh

"l"P'h .,1. OMe

OH

O

2.8.13

0

Ph 2.4.4

2.1.5

O

vi

OMe

OH

2.1.3

2.8.11

O

i v, v

=

xi

~~-,H CO2Me

Ph, H

2.1.5

2.4.5

Conditions: (i)Mucor miehei lipase MAP-10, i-BuOH, 2.8.10 (42%, 95% e.e.), 2.8.11 (43%, 95%

e.e.); (ii) Et2NH2Br, Et2A1C1, CH2C12,-15 ~ 90%; (iii) NAN3, DMF, 65-70 ~ 80%; (iv) PhCOC1, DMAP, CH2C12, rt, 93%; (v) H2, Pd/C, 50 psi, 80%; (vi) NAN3, aq. acetone, NH4C1, refl., 95%; (vii) PhCOC1, DMAP, CH2C12, rt, 91%; (viii) H2, Pd/C, 50 psi, 93%; (ix) Na2CO3, aq. MeOH, rt, then CH2N2, 81%; (x) SOC12, CHC13, reflux, 73%; (xi) 1 N HC1, MeOH, 80%.

281

[Scheme 30 ~1

Ph 2.1.11

H-_,,,~~_-CO2Me Ph H

H_,,,~z~-~-CO2H Ph H 2.8.14

2.4.5

\ AcO

Ph 0

O OSiEt3 iii

Taxol,

O....
0.1

3.6.16

Bz

PhOCH2

5.81

>0.1

3.6.17

t-BuO

4-thiazolyl

1.36

0.0014

3.6.18

t-BuO

methyl

1.08

0.043

3.6.19

t-BuO

3.6.20

t-BuO

vinyl ethyl

0.92 0.61

0.016 0.011

3.6.21

t-BuO

butyl

2.15

0.018

3.6.22

t-BuO

cyclohexyl

0.57

0.014

3.6.23

t-BuO

isobutyl

0.95

0.00036

3.6.24

t-BuO

benzyl

>17

>0.1

Cpd

IC50 (~g/mL) b P388

3.6.25 t-BuO pentyl 2.0 0.016 (a) ID50=Drug concentration that reduces tubulin concentration by 50%. (b) see Table 6.

293 The activity of the 3'-alkyl derivatives of Taxotere @ (in particular isobutyl analog 3.6.23 and cyclohexyl derivative 3.6.22) is especially remarkable. The authors briefly discuss the superior performance of these compounds in vivo [76] and comment that they are less toxic than Taxol| The authors also conclude that the C-3' substituent must fit within a hydrophobic pocket, and that this pocket is obviously limited in size (for example, the 3' benzyl derivative 3.6.24 is totally inactive). 6.3.7. Extended Side Chain Analogs I n t e r e s t i n g analogs of Taxol| and Taxotere (!i) with one carbonhomologated side chains were synthesized by Georg and co-workers. The compounds were p r e p a r e d using 10-DAB and Commerqon's modified oxazolidineacetic acid protocol. Unfortunately, neither 3.7.1 nor 3.7.2 (Figure 4) displayed any significant activity in a tubulin assembly assay. The lack of activity was attributed to unfavorable conformations of these analogs which might prevent binding to the receptor site on microtubules [77]. In addition, as discussed in section 6.3.6 (see 3.6.24, Table 7) there seems to be a well-defined limitation to the length of the side chain that can achieve proper binding, and addition of an extra methylene unit to such chain is totally deleterious here as well. O

AcO

O OH

R-~NH -

O .... OH

O

O

3.7.1. R = Ph 3.7.2. R = t-BuO Figure 4: Extended Side Chain Analogs [77] 6.4. CONFORMATIONAL STUDIRS Because the topology of the binding site on the microtubules and the bound state conformation of Taxol |

are unknown, potentially important

294 features of the binding site have been derived from the reported crystal structure of Taxotere | 2.1.29 [78]. The solution conformation of Taxol | was studied by NMR spectroscopy in non-aqueous solvents and it was found to be very similar to the one observed in the crystal structure of Taxotere | NOESY and ROESY experiments suggested that the side chain is folded under the diterpene core; this was further supported by NOEs observed between H-2' and the C-4-acetyl group. The observed small JH2'-H3' coupling constant of 2.7 Hz is indicative of hindered rotation within the side chain, which is attributed to the intramolecular hydrogen bonding [79-82]. Swindell and collaborators reported conformational analysis of methyl N-benzoylisoserinate (2.1.5)and methyl phenyllactate using MM2 calculations. These modeling studies suggested that the hydrogen bonding or electrostatic interactions involving the proximate C=O, OH, and NH moieties in the side chains are the major determinants of side chain conformation [59]. Gu~ritteVoegelein studied the conformation of Taxotere | and some side chain analogs and proposed a similar intramolecular hydrogen bonding [60]. Swindell proposed that these interactions "preorganize" the side chain for binding to microtubules [59]. However, recent NMR studies of Taxol| and Taxotere | in polar solvents (DMSO/H20 and MeOH) suggest a different side chain arrangement. It was proposed t h a t Taxotere | and all 2'R, 3'S active analogs display conformations in which the C-2 benzoate group holds the side chain in a particular spatial arrangement. This new conformation is stabilized by a hydrophobic interaction between the C-2 benzoate and the C-3' N-benzoyl or NBoc groups. Thus, hydrophobic interactions together with the network of intramolecular hydrogen bonding discussed above would be responsible for a specific orientation of the phenylisoserine side chain. This study also led the authors to suggest a binding process of active taxanes to tubulin: the first step is the recognition of the taxane core by the binding site; the second step is hydrophobic interactions between the C-2 benzoate and the C-3' amino substituents of the side chain. These interactions orient the side chain in such a position that the C-2' hydroxyl and the C-3' phenyl groups can interact with appropriate tubulin residues, leading to stabilization of the drug-receptor complex [83]. Scott and Swindell studied conformations of Taxol|

and its side chain

methyl ester 2.1.5 by NMR spectroscopy and molecular modeling in an

295 aqueous environment. Interestingly, for the side chain methyl ester 2.1.5, the coupling constant JH2'-H3' changed from 2.1 Hz in chloroform to 4.7 Hz in a 1:1 w ater/DMSO-d6. The conformational equilibrium of the methyl ester thus shifted from a gauche to an anti conformer. Similarly, JH2'-H3' for Taxol| also changed from 2.7 (CDC13) to 8 Hz in a mixture of water and DMSO; this points to the dominant contribution of conformers with large torsional angle and indicative of a different side chain conformational ensemble.

Molecular modeling studies found four low-energy conformations

for Taxol| in three of them, the 2' and 3' protons were in g a u c h e arrangement, similar to the crystal structure; the fourth conformer favored the anti arrangement and was the dominant Taxol| conformation in aqueous solution [84]. The Kansas group reported new NMR studies on Taxol| and Taxotere | in support of their "hydrophobic collapse" theory. These studies suggest that water strongly induces the same type of conformation in both Taxol| and Taxotere| the key interaction is the hydrophobic clustering of the 2-benzoyl, 3'-phenyl, and 4-acetyl groups; the N-benzoyl or Boc groups are not participating in organizing the side chain. The NOESY and ROESY experiments on Taxotere | in a 1:1 mixture of methanol/water and Taxol | in DMSO/H20 displayed cross peaks between the aromatic signals on the different rings. Based on the theory that the chemical shift changes of the signals arise from mutual ring-current effects, these new data demonstrate the closeness of these aromatic rings. The strongest interactions were observed between the meta and para protons of the 3'-phenyl and the ortho and meta protons of the C-2 benzoyl group; and furthermore, no cross peaks were observed between t-butyl of the N-Boc and C-2 benzoyl groups

[85]. Since no active Taxol|

analogs are known with deleted 2-benzoyl and 3'-

phenyl groups, it is quite likely that these groups are providing the putative "preorganization" of the side chain conformation most relevant to binding, via hydrophobic clustering rather than intramolecular hydrogen bonding. Structural information on the microtubule binding site and its complex with the drug is needed to understand more about the active conformation of Taxol |

and its tubulin polymerization mechanism.

296 RE~'EtCENCES ~

~

3. 4. 5. ~

o

o

9. 10. 1 1 ~

12. 13. 14. 15. 16.

.

Wani, M.C.; Taylor, H.L.; Wall, M.E.; Coggon, P.; McPhail, A.T.J. Am. Chem. Soc. 1971, 93, 2325. Rowinsky, E.K.; Donehower, R.C. Pharmacol. Ther. 1991, 52, 35. Rowinsky, E.K.; Donehower, R.C.J. Natl. Cancer Inst. 1991, 83, 1778. Horwitz, S.B. Trends Pharm. Sci. 1992, 13, 134. For a recent account: Nicolaou, K.C.; Dai, W-M.; Guy, R.D. Angew. Chem. Int. Ed. Engl. 1994, 33, 15. (a) Holton, R.A.; Somoza, C.; Kim, H-B.; Liang, F.; Biediger, R.J.; Boatman, P.D.; Shindo, M.; Smith, C.C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, K.K.; Gentile, L.N.; Liu, J.H.J. Am. Chem. Soc. 1994, 116, 1597. (b). Holton, R.A.; Somoza, C.; Kim, H-B.; Liang, F.; Biediger, R.J.; Boatman, P.D.; Shindo, M.; Smith, C.C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, K.K.; Gentile, L.N.; Liu, J . H . J . Am. Chem. Soc. 1994, 116, 1599. Nicolaou, K.C.; Yang, Z.; Liu, J.J.; Ueno, H.; Nantermet, P.G.; Guy, R.K.; Claiborne, C.F.; Renaud, J.; Couladouros, E.A.; Paulvannan, K.; Sorensen, E.J. Nature 1994, 367, 630. Cragg, G.M.; Snader, K.M. Cancer Cells 1991, 259, 181. Bormann, S. Chem. Eng. News 1992, 7 0 , 3 0 . Denis, J.-N.; Greene, A.E.; Gu~nard, D.; Gu~ritte-Voegelein, F.; Mangatal, L.; Potier, P. J. Am. Chem. Soc. 1988, 110, 5917. Chauvi~re, G.; Gu~nard, D.; Picot, F.; S~nilh, V.; Potier, P. C.R. Seances Acad. Sci., Ser. 2 1981, 293, 501. Kingston, D.G.I. Pharmacol. Ther. 1991, 52, 1. Swindell, C.S. Org. Prep. Proc. Int. 1991, 4. 465. Gu~nard, D.; Gu~ritte-Voegelein, F.; Potier, P. Acc. Chem. Res. 1993, 26, 160. Suffness, M. Ann. Rep. Med. Chem. 1993, 28, 305. Kingston, D.G.I.; Molinero, A.A.; Rimoldi, J.M. in Progress in the Chemistry of Organic Natural Products; Herz, W., Kirby, G.W., Moore,

297

17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

R.E., Steglich, W., Tamm, C., Eds.; Springer: New York, 1993; vol. 61; p.1. Georg, G.I.; Ali, S.M.; Zygmunt, J.; Jayasinghe, L.R. Exp. Opin. Ther. Pat. 1994, 4, 109. Boa, A.N.; Jenkins, P.R.; Lawrence, N.J. Cont. Org. Synth. 1994, 1, 47. Georg, G.I.; Boge, T.C.; Cheruvallath, Z.S.; Clowers, J.S.; Harriman, G.C.B.; Hepperle, M.; Park, H. in Taxol: Science and Applications; Suffness, M., Ed.; CRC: Boca Raton (in press). Denis, J.-N.; Greene, A.E.; Serra, A.A.; Luche, M-J. J. Org. Chem. 1986, 51, 46. Denis, J.-N.; Correa, A.; Greene, A.E.J. Org. Chem. 1990, 55, 1957. Commerqon, A.; B~zard, D.; Bernard, F.; Bourzat, J.D. Tetrahedron Lett. 1992, 33, 5185. Denis, J.-N.; Kanazawa, A.M.; Greene, A.E. Tetrahedron Lett. 1994, 35, 105. Didier, E.; Fouque, E.; Commerqon, A. Tetrahedron Lett. 1994, 35, 3063. Deng, L.; Jacobsen, E . N . J . Org. Chem. 1992, 57, 4320. Wang, Z-M.; Kolb, H.C.; Sharpless, K.B.J. Org. Chem. 1994, 59, 5104. Koskinen, A.M.P.; Karvinen, E.K.; Siiril~i, J . P . J . Chem. Soc. Chem. Commun. 1994, 21. Mangatal, L.; Adeline, M.-T.; Gu~nard, D.; Gu~ritte-Voegelein, F.; Potier, P. Tetrahedron 1989, 45, 4177. Denis, J.-N.; Correa A.; Greene, A . E . J . Org. Chem. 1991, 56, 6939. Kanazawa, A.M.; Correa, A.; Denis, J.-N.; Luche, M.-J.; Greene, A.E. J. Org. Chem. 1993, 58, 255. Bunnage, M. E.; Davies, S. G.; Goodwin, C. J. J. Chem. Soc. Perkin Trans. 1 1994, 2385. Palomo, C.; Arrieta, A.; Cossio, F.P.; Aizpurua, J.M.; Mielgo, A.; Aurrekoetxea, N. Tetrahedron Lett. 1990, 31, 6429. Cainelli, G.; Panunzio, M.; Giacomini, D.; Di Simone B.; Camerini, R. Synthesis 1994, 805. (a) Holton, R.A. European Patent 0 400 971, 1990. (b) Holton, R.A.U.S. Patent 5 015 744, 1991. (c) Holton, R. A. Abstracts of Papers, 203rd National Meeting of the American Chemical Society, San Francisco, CA, American Chemical Society: Washington, DC, 1992; ORGN 0355. (d) Holton, R.A.U.S. Patent 5 175 315, 1992.

298 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56.

Ojima, I.; Habus, I.; Zhao, M.; Georg, G.I.; Jayasinghe, L.R.J. Org. Chem. 1991, 56, 1681. Ojima, I.; Habus, I.; Zhao, M.; Zucco, M.; Park, Y.H.; Sun, C.M.; Brigaud, T. Tetrahedron 1992, 48, 6985. Ojima, I.; Zucco, M.; Duclos, O.; Kuduk, S.D.; Sun, C.M.; Park, Y.H. Bioorg. Med. Chem. Lett. 1993, 3, 2479. Georg, G.I.; Cheruvallath, Z.S.; Harriman, G.C.B.; Hepperle, M.; Park, H. Bioorg. Med. Chem. Lett. 1993, 11, 2467. Georg, G.I.; Mashava, P.M.; Akgtin, E.; Milstead, M. Tetrahedron Lett. 1991, 32, 3151. Georg, G.I.; Akgtin, E.; Mashava, P.M.; Milstead, M.; Ping, H.; Wu, Z.; Vander Velde, D.; Takusagawa, F. Tetrahedron Lett. 1992, 33, 2111. Farina, V.; Hauck, S. I.; Walker, D. G. Synlett. 1992, 57, 4320. Bourzat, J.D.; Commer~on, A. Tetrahedron Lett. 1993, 34, 6049. Holton, R.A.; Liu, J.H. Bioorg. Med. Chem. Lett. 1993, 3, 2475. Rey, A.W.; Droghini, R.; Douglas, J.; Vemishetti, P.; Boettger, S. D.; Racha, S.; Dillon, J. L. Can. J. Chem. 1994, 72, 2131. Davis, F.A.; Reddy, R.T.; Reddy, R.E.J. Org. Chem. 1992, 57, 6387. Mukai, C.; Kim, I. J.; Furu, E.; Hanaoka, M. Tetrahedron 1993, 49, 8323. Mukai, C.; Kim, I. J.; Furu, E.; Hanaoka, M. Tetrahedron Asymmetry 1992, 3, 1007. Hattori, K.; Yamamoto, H. Tetrahedron 1994, 50, 2785. Swindell, C.S.; Tao, M. J. Org. Chem. 1993, 58, 5889. Kanazawa, A.M.; Denis, J.-N.; Greene, A.E.J. Org. Chem. 1994, 59, 1238. HSnig, H.; Seufer-Wasserthal, P.; Weber, H. Tetrahedron 1990, 46, 3841. Brieva, R.; Crich, J. Z.; Sih, C.J.J. Org. Chem. 1993, 58, 1068. Patel, R.N.; Banerjee, A.; Ko, R.Y.; Howell, J.M.; Li, W.-S.; Comezoglu, F.T.; Partyka, R.A.; Szarka, L. Biotechnol. Appl. Biochem. 1994, 20, 23. Gou, D.-M.; Liu, Y.-C.; Chen, C.-S. J. Org. Chem. 1993, 58, 1287. Kingston, D.G.I.; Chaudhary, A.G.; Gunatilaka, L.A.A.; Middleton, M.L. Tetrahedron Lett. 1994, 35, 4483. Patel, R.N.; Banerjee, A.; Howell, J.M.; McNamee, C.G.; Brozozowski, D.; Mirfakhrae, D.; Nanduri, V.; Thottathil, J.K.; Szarka, L. Tetrahedron Asymmetry 1993, 4, 2069.

299 57. 58. 59. 60. 61. 62. 63. 4.

65.

66. 67. 68.

69. 70. 71.

Kearns, J.; Kayser, M.M. Tetrahedron Lett. 1994, 35, 2845. Parness, J.; Kingston, D.G.I.; Powell, R.G.; Harracksingh, C.; Horwitz, S.B. Biochem. Biophys. Res. Commun. 1982, 105, 1082. Swindell, C.S.; Krauss, N.E.; Horwitz, S.B.; Ringel, I. J. Med. Chem. 1991, 34, 1176. Gu~ritte-Voegelein, F.; Gu~nard, D.; Lavelle, F.; Le Goff, M-T.; Mangatal, L.; Potier, P. J. Med. Chem. 1991, 34, 992. Ringel, I.; Horwitz, S.B.J. Natl. Cancer Inst. 1991, 83,288. Bissery, M.-C.; Gu~nard, D.; Gu~ritte-Voegelein, F.; Lavelle, F. Cancer Res. 1991, 51, 4845. Georg, G.I.; Cheruvallath, Z.S.; Himes, R.H.; Mejillano, M.R. Bioorg. Med. Chem. Lett. 1992, 2, 1751. Georg, G.I.; Boge, T.C.; Cheruvallath, Z.S.; Harriman, G.C.B.; Hepperle, M.; Park, H.; Himes, R.H. Bioorg. Med. Chem. Lett. 1994, 4, 335. Georg, G.I.; Boge, T.C.; Cheruvallath, Z.S.; Harriman, G.C.B.; Hepperle, M.; Park, H.; Himes, R.H. Bioorg. Med. Chem. Lett. 1994, 4, 1825. Georg, G.I.; Cheruvallath, Z.S.; Himes, R.H.; Mejillano, M.R.; Burke, C.T.J. Med. Chem. 1992, 35, 4230. Swindell, C.S.; Heerding, J.M.; Krauss, N.; Horwitz, S.B.; Ringel, I. Bioorg. Med. Chem. Lett. 1994, 4, 1531. Georg, G.I.; Boge, T.C.; Cheruvallath, Z.S.; Harriman, G.C.B.; Hepperle, M.; Park, H.; Himes, R.H. Bioorg. Med. Chem. Lett. 1994, 4, 2331. Georg, G.I.; Harriman, G.C.B.; Hepperle, M.; Himes, R.H. Bioorg. Med. Chem. Lett. 1994, 4, 1381. Boge, T.C.; Himes, R.H.; Vander Velde, D.G.; Georg, G.I.J. Med. Chem. 1994. 37, 3337. Ojima, I.; Park, Y.H.; Fenoglio, I.; Duclos, O.; Sun, C-M.; Kuduk, S.D.; Zucco, M.; Appendino, G.; Pera, P.; Veith, J.M.; Bernacki, R.J.; Bissery, M.-C.; Combeau, C.; Vrignaud, P.; Riou, J.F.; Lavelle, F. In Taxanes Anticancer Agents: Basic Science and Current Status; Georg, G.I.; Chen, T.T.; Ojima, I.; Vyas, D.M. Eds.; ACS Symp. Series, American Chemical Society, Washington, D.C., 1995, p. 262.

300 72. 73. 74. 75. 76.

77. 78. 79. 80. 81. 82. 83. 4~

85.

Ojima, I.; Duclos, O.; Kuduk, S.D.; Sun, C-M.; Slater, J.C.; Lavelle, F.; Veith, J.M.; Bernacki, R.J. Bioorg. Med. Chem. Lett. 1994, 4, 2631. Ojima, I.; Duclos, O.; Zucco, M.; Bissery, M.-C.; Combeau, C.; Vrignaud, P.; Riou, J.F.; Lavelle, F. J. Med. Chem. 1994, 37, 2603. Kant, J.; Huang, S.; Wong, H.; Fairchild, C.; Vyas, D.; Farina, V. Bioorg. Med. Chem. Lett. 1993, 3, 2471. Maring, C.J.; Grampovnik, D.J.; Yeung, C.M.; Klein, L.L.; Li, L.; Thomas, S.A.; Plattner, J.J. Bioorg. Med. Chem. Lett. 1994, 4, 1429. Li, L.; Thomas, S.A.; Klein, L.L.; Yeung, C.M.; Maring, C.J.; Grampovnik, D.J.; Lartey, P.A.; Plattner, J.J.J. Med. Chem. 1994, 37, 2655. Jayasinghe, L.R.; Datta, A.; Ali, S.M.; Zygmunt, J.; Vander Velde, D.G.; Georg, G.I.J. Med. Chem. 1994, 37, 2981. Gu6ritte-Voegelein, F.; Mangatal, L. Gu6nard, D.; Potier, P.; Guilheim, J.; Cesario, M.; Pascard, C. Acta CrystaUogr. 1990, C46, 781. Chmurny, G.N.; Hilton, B.D.; Brobst, S.; Look, S.A.; Witherup, K.M.; Beutler, J.A.J. Nat. Prod. 1992, 55, 414. Hilton, B.D.; Chmurny, G.N.; Muschik, G.M.J. Nat. Prod. 1992, 34, 1176. Baker, J.K. Spectroscopy Letters, 1992, 25, 31. Falzone, C.J.; Benesi, A.J.; Lecomte, J.T.J. Tetrahedron Lett. 1992, 33, 1169. Dubois, J.; Gu4nard, D.; Gu4ritte-Voegelein, F.; Guedira, N.; Potier, P.; Gillet, B.; Beloeil, J.-C. Tetrahedron 1993, 49, 6533. Williams, H.J.; Scott, I.A.; Dieden, R.A.; Swindell, C.S.; Chirlian, L.E.; Francl, M.M.; Heerding, J.M.; Krauss, N.E. Tetrahedron 1993, 49, 6545. Vander Velde, D.G.; Georg, G.I.; Grunewald, G.L.; Gunn, C.W.; Mitscher, L.A.J. Am. Chem. Soc. 1993, 115, 11650.

The Chemistry and Pharmacology of Taxol and its Derivatives V. Farina, editor 9 1995 Elsevier Science B.V. All rights reserved

301

7 THE BIOCHEMICAL P H A R M A C O L O G Y OF T A X O L | A N D M E C H A N I S M S OF RESISTANCE Lisa M. Landino and Timothy L. Macdonald Department of Chemistry, University of Virginia, Charlottesville, VA 22901, U.S.A.

7.1. INTRODUCTION Microtubules, like DNA and RNA, are conserved structures in all eukaryotic cells with numerous and diverse functions. By forming a scaffolding network within cells, microtubules maintain cell shape. In neurons, microtubules form a track through the axons along which organelles and proteins are transported. In addition, microtubules are the primary components of the mitotic spindle which forms during cell division. All eukaryotic cells utilize a spindle, composed of microtubules, to segregate chromosomes during mitosis [1]. The principal component of microtubules is the protein tubulin, a heterodimer composed of two similar subunits, (~ and ~-tubulin. The molecular weight of each subunit is approximately 50 kDa. Sequence analysis of the major a - a n d ~-tubulin gene products expressed in mammalian cells shows about 40% homology between the two tubulins [2]. The ability to reversibly assemble into microtubules (polymer) and

302 disassemble to the tubulin heterodimer (monomer) is an intrinsic property of t h i s d i m e r i c protein. A l t e r n a t i n g a - a n d ~-tubulins assemble longitudinally into protofilaments which t h e n join l a t e r a l l y w i t h other protofilaments to form the cylindrical microtubule s t r u c t u r e . A typical microtubule is composed of 13 protofilaments with an outer d i a m e t e r of 28 nm and an inner diameter of 14 n m [1] (Figure 1).

Figure 1. Assembly of tubulin monomers into a microtubule structure.

303 In addition, a number of aberrant structures can be formed in vitro including rings and sheets. Purified tubulin can be assembled in vitro at 37~ in the presence of GTP, Mg 2+, and a calcium chelator [3]. Two molecules of GTP bind to each a,~ heterodimer and, upon incorporation into the microtubule polymer, one molecule of GTP is hydrolyzed to GDP and phosphate. In vivo, r e g u l a t i o n of microtubule a s s e m b l y and disassembly is mediated by microtubule-associated proteins (MAPs) and fluctuations in intracellular calcium concentrations [4]. The numerous and diverse functions of microtubules require facile, yet regulated, assembly and disassembly in response to intracellular stimuli. MAPs are thought to be principally associated with tubulin through charge interactions at specific sites along the microtubule polymer and these interactions can be modulated by intracellular signaling events such as MAP phosphorylation [5]. MAPs are a large and diverse family of proteins which co-purify with tubulin through in vitro cycles of assembly and disassembly. Ion exchange chromatography separates the negatively charged tubulin from the more basic MAPs [6]. Only a handful of the most abundant MAPs, such as MAP1, MAP2, and tau, have been characterized in any detail, although many other minor proteins associate with tubulin in vivo. The precise roles of these associated proteins is of considerable interest since the regulation of microtubule function is critical to cell growth and viability. The process of tubulin polymerization to microtubule structures can be dissected into discrete steps. Tubulin polymerization requires a slow nucleation phase, followed by rapid elongation of the microtubule structure, and ultimately an equilibrium p l a t e a u is reached. The f i l a m e n t o u s microtubule structures are in equilibrium with a pool of unpolymerized (z,~ heterodimers both in vivo and in vitro. The concentration of unpolymerized tubulin monomer at this steady state (in equilibrium with polymer) is referred to as the critical concentration (Cc). At tubulin concentrations below the critical concentration, microtubules will not form. A number of factors influence the Cc in vitro, including t e m p e r a t u r e , pH, and the presence of MAPs or drugs such as colchicine or Taxol| [7,8]. In 1981, Carlier and Pantaloni had observed that GTP hydrolysis was not coupled to tubulin polymerization, but that it occurred after monomer incorporation [9]. Their investigations suggested that GTP hydrolysis was

304 not a driving force in the assembly of microtubules, but r a t h e r hydrolysis of GTP-bound t u b u l i n m o n o m e r s in the assembled microtubule induces a conformational

change

in

the

microtubule

structure.

Such

a

conformational role of nucleotide t r i p h o s p h a t e hydrolysis (both ATP and GTP) has emerged as a fairly common p h e n o m e n o n in biological systems [10]. Investigation of the interactions of any tubulin-specific compound with t u b u l i n a n d m i c r o t u b u l e s r e q u i r e s an u n d e r s t a n d i n g of the dynamic properties of microtubules [11,12].

As a dynamic s t r u c t u r e , there is a

constant flux between the monomeric and polymeric forms.

If the rates of

m o n o m e r addition and loss at the microtubule ends are identical, no change in microtubule length at equilibrium is detected. While GTP-bound (z,~ heterodimers are incorporated into the plus (+) or assembly end of the microtubule, GDP-bound tubulin heterodimers are lost from the minus (-) or disassembly end of the microtubule.

This dynamic property of flux or

t r e a d m i l l i n g is observed in vitro using purified tubulin; however, the presence of MAPs can suppress this dynamic behavior [13] (Figure 2).

(+) ASSEMBLY

(-) DISASSEMBLY MICROTUBULE AT EQUILIBRIUM % (~0

Figure 2. Microtubules have distinct assembly (+) and disassembly (-) ends. At equilibrium, the rates of monomer addition and loss are constant and no net change in microtubule length is observed. This dynamic behavior, known as treadmilling or flux, can be suppressed by antimitotic drugs such as vinblastine or Taxol| Another dynamic property of microtubules is dynamic instability [14]. An individual microtubule can undergo t r a n s i t i o n s of l e n g t h e n i n g and s h o r t e n i n g which are observable both in vitro and in vivo.

The growth

305 phase is stabilized by a GTP-cap which exists at high t u b u l i n concentrations, since the rate of monomer addition will transiently exceed the rate of GTP hydrolysis. However, if GTP-bound tubulin concentrations are low, the rate of GTP hydrolysis will exceed the rate of monomer addition. The stabilizing GTP-cap at the assembly end (+) will be lost and this will induce rapid depolymerization. This dynamic property can also be suppressed by the presence of MAPs [15]. Suppression of the dynamic properties of microtubules by antimitotic agents has been suggested as a plausible mechanism of cytotoxicity. A number of in vitro and in vivo studies by J o r d a n and Wilson have d e m o n s t r a t e d t h a t microtubule dynamics can be s u p p r e s s e d by substoichiometric concentrations of vinblastine and Taxol| [16-21]. The results of their studies and the mechanistic implications of their findings are presented in section 7.2. 7.2. THE T U B U L I N / M I C R O T t ~ U I ~

SYSTEM AS A D R U G TARGET

The tubulin/microtubule system has captured the a t t e n t i o n of medicinal chemists since it is the target of a number of synthetic molecules and natural products including colchicine, vinblastine, and Taxol | [1, 7, 8]. The structures of these natural products, as well as the semi-synthetic Taxol| derivative, Taxotere | are shown in Figure 3. The therapeutic utility of compounds which interact with tubulin and microtubules results initially from their ability to disrupt normal spindle function. Such compounds are often called spindle poisons or antimitotic agents. The compounds t h a t bind to tubulin are chemically divergent and there is evidence that they bind to the ~-subunit of the tubulin heterodimer [22, 23]. Colchicine and vinblastine bind to the unpolymerized tubulin monomer at two distinct sites, as demonstrated by competitive binding assays [7, 8]. Monomer binding shifts the monomer-polymer equilibrium toward the depolymerized state. The majority of compounds which bind to tubulin inhibit its polymerization in vitro. The exception is the diterpenoid Taxol| isolated from the bark of the Pacific yew tree, Taxus brevifolia. Taxol| has received much attention due to its antitumor activity, its unique s t r u c t u r e and m e c h a n i s m of action. Unlike colchicine and vinblastine, which inhibit tubulin polymerization, Taxol | stabilizes

306 microtubules and shifts the equilibrium toward the polymerized state. This novel mode of action sets Taxol |

apart and has sparked a renewed

interest in the tubulin/microtubule system as a chemotherapeutic target.

H•O

eee##J

Me0 . ~ ~.~...~ 'NHAc

Ac

MeO

HMeO~N~OH ! Me

~

0 OMe

Vinblastine (E=COOMe)

Colchicine

O

R1"~NH

0

Phil_

R20~, 20H o .... _

OH

0

.o

Taxol, RI=Ph; R2=Ac Taxotere, Rl=t-BuO; R2=H Figure 3. Structures of the most important antimitotic agents The molecular pharmacology of Taxol | has been examined in detail by Horwitz and coworkers [24-28]. These researchers demonstrated that this drug preferentially binds to the microtubule polymer rather than to the tubulin monomer. Taxol | binds reversibly to microtubules with a binding constant of -1 ~M. In addition, the Taxol | binding site is distinct from the sites occupied by GTP, colchicine, and vinblastine.

The microtubules

formed in the presence of Taxol | in vitro are extremely stable and have properties distinct from normal microtubules. Taxol | induces tubulin polymerization in the absence of GTP or MAPs and the microtubules are resistant to depolymerization induced by calcium ion. Taxol |

will induce

307 tubulin polymerization at low temperatures (4 ~ but, in this case, GTP or MAPs are required for maximal polymer formation [24]. A number of in vitro studies have been undertaken to investigate the interaction of Taxol| with tubulin and microtubules at both the kinetic and thermodynamic level. Since tubulin has not been crystallized, the specific molecular i n t e r a c t i o n s of antimitotic agents with the protein have remained elusive. However, a variety of biophysical methods have been employed which have provided much insight into tubulin-drug interactions [29]. In addition, since colchicine, vinblastine, and Taxol| bind at distinct sites, the thermodynamic relationships between these ligand binding sites and their effect on microtubule assembly can be addressed. Timasheff and coworkers have examined the combined effects of Taxol| and colchicine on the thermodynamics of tubulin polymerization in vitro [30]. The stabilizing effect of Taxol | is capable of overriding the destabilizing effect of agents such as colchicine and vinblastine. However, the stabilizing free energy for addition of each a,~ heterodimer gained upon binding of Taxol | is diminished for the tubulin-colchicine complex vs. pure tubulin. The binding of Taxol | provides more t h a n - 3 . 0 kcal/mol of free energy in the polymerization of pure tubulin into microtubules, whereas only -0.5 kcal/mol is gained for Taxol| polymerization of the tubulin-colchicine complex. This reduction in stabilizing free energy implies that energy is expended to overcome unfavorable factors such as sterics or geometric strain in the tubulin-colchicine complex. In addition, Taxol| polymerization of the tubulin-colchicine complex required both h e a t and GTP, whereas pure t u b u l i n would assemble at 10 ~ in the absence of GTP. The processes of tubulin polymerization and GTP binding and hydrolysis are linked under normal polymerization conditions. Essentially, the energy imparted by Taxol | binding to the microtubule decreases the cooperativity of these processes and, as a result, Taxol| tubulin polymerization does not require GTP binding or hydrolysis. In addition, Carlier and Pantaloni showed that Taxol| does not interfere with GTP binding or hydrolysis at the exchangeable GTP binding site [31]. Although Taxol| can induce polymerization of GDP-bound tubulin monomers, it is important to note that, if GTP is present, hydrolysis does occur.

308 Based on this thermodynamic analysis, the action of Taxol |

may be

exerted on the lateral interactions between protofilaments which are normally weak. Differences in the conformation of the tubulin-colchicine complex vs. pure tubulin at these lateral junctions would translate into different l i g a n d b i n d i n g s t r e n g t h s . Careful c o n s i d e r a t i o n of the thermodynamics of tubulin-ligand binding may provide insight into the in v i v o mechanism of these antimitotic agents. The selectivity of Taxol (ii) against particular tumor cell lines may stem from its ability to contribute maximal binding energy under specific physiological conditions. The presence of specific MAPs, for example, may dictate the thermodynamic consequences of Taxol | binding. Some of the most convincing evidence to explain the mechanism of antimitotic agents comes from the work of Jordan and Wilson [15-21, 32-34]. Early and simplistic models to explain the cellular m e c h a n i s m of colchicine and vinblastine suggested t h a t these compounds disrupted cellular function by preventing microtubule formation. Subsequent in vivo studies revealed that, at therapeutically useful concentrations of these depolymerizing agents, microtubules were still formed. In the case of t h e r a p e u t i c a l l y useful levels of Taxol (ii), all cellular tubulin was not polymerized, suggesting that antimitotic agents interact with their cellular target in a more subtle, but intriguing manner. The continuing efforts of these researchers have examined the interactions of antimitotic agents with microtubule ends and surfaces and have revealed how alterations in microtubule dynamics can modulate cellular function and viability. The o b s e r v a t i o n t h a t T a x o l | m i c r o t u b u l e s do not depolymerize significantly when diluted or cooled suggests t h a t the dissociation rate constant of tubulin monomer is decreased by Taxol | binding to the microtubule. Investigation of the dynamics of Taxol | stabilized microtubules has d e m o n s t r a t e d such a reduction in the dissociation rate constant of tubulin from microtubules [32]. The two dynamic properties of microtubules which have been investigated most thoroughly are dynamic instability and treadmilling. The dynamic behavior known as dynamic instability, in which an individual microtubule undergoes periods of growing and shortening, is not as well understood as treadmilling. However, on a simplistic level, the importance of such a behavior during mitosis and chromosomal segregation can be appreciated.

309 Individual

microtubules

must

grow

and

shorten

in

response

to

intracellular stimuli in order for cell division to occur. The dynamic instability of microtubules is influenced by the presence of microtubuleassociated proteins (MAPs). Although MAPs will suppress this behavior in vitro, it can still occur to a detectable extent in bovine brain microtubules, which contain approximately 70% tubulin and 30% MAPs [20].

Like

dynamic instability, treadmilling is also suppressed by MAPs [21]. The net addition of GTP-bound monomers and loss of GDP-bound monomers at the microtubule ends occurs at the polymer steady state. This equilibrium phenomenon has been demonstrated in vitro; in addition, mitotic spindle microtubules, which are very dynamic st ruct ures, display the same behavior. Agents such as v i n b l a s t i n e

and colchicine i n h i b i t microtubule

assembly at substoichometric concentrations in vitro. vinblastine

to d i s r u p t

microtubule

formation

at

The ability of

a ratio

of 300:1

(tubulin:vinblastine) supports a mechanism whereby the drug binds to the ends of microtubules, not to soluble tubulin monomer [33]. Wilson et al. have also d e m o n s t r a t e d t h a t tubulin exchange at microtubule ends is inhibited by 50% when only -1.2 molecules of vinblastine are bound per microtubule [34]. Wilson and coworkers propose t h a t s u p p r e s s i o n of microtubule dynamics at low concentrations of vinblastine correlates with mitotic block at the m e t a p h a s e / a n a p h a s e boundary.

In addition to suppression of

microtubule dynamics, vinblastine also enhances the length of time that a microtubule spends in an a t t e n u a t e d state in which no growing or shortening of the microtubule is detected. Notably, Wilson's laboratory has demonstrated t h a t low vinblastine concentrations did not prevent mitotic spindle formation, but still blocked HeLa cells at metaphase.

The mitotic

spindle was fully formed in the proper bipolar configuration; however, the alterations induced by low vinblastine concentrations were much more subtle

as

determined

by

immunofluorescence

microscopy.

These

alterations were sufficient to prevent the transition from metaphase to anaphase.

The overall structure of the microtubules and the kinetochore

was not affected by the drug, but the number of microtubule-kinetochore attachments was decreased. Microtubule-kinetochore attachment is vital to chromosomal segregation during the later stages of mitosis.

310 The concentrations

of vinblastine

which induced

these

subtle

alterations in the mitotic spindle, without inducing depolymerization of the microtubules, were in the low nanomolar range (0.1 - 6 nM). Following incubation with 2 nM vinblastine for 18-20 hours, no HeLa cells were detected in anaphase for 2 - 48 hours after treatment with the drug. This indicates that mitotic block at the metaphase/anaphase boundary is not transient. In addition, a substantial percentage (31-38%) of cells in interphase treated with low nanomolar Taxol| concentrations (3-10 nM) had multiple nuclei. Jordan et al. examined the mechanism of mitotic block and inhibition of cell proliferation by low concentrations of Taxol| [16]. Despite their seemingly opposite mechanisms of action, Taxol| and vinblastine demonstrate nearly identical effects on the mitotic spindle in HeLa cells. Like vinblastine, Taxol|

induced mitotic block at the metaphase/anaphase

boundary. The concentration of Taxol| which induced 50% mitotic block in HeLa cells was only 8 nM. This concentration of Taxol | which resulted in significant mitotic arrest did not increase microtubule mass in vivo as had been anticipated for this microtubule-stabilizing agent. The effect of low Taxol | concentrations on mitotic spindle organization was also determined. Immunofluorescence microscopy revealed t h a t spindles which had been treated with nanomolar levels of Taxol| were nearly identical to spindles formed in the presence of low concentrations of other antimitotic agents including vinblastine.

Astral microtubules were more

obvious in Taxol| cells when compared to the control cells. This is in agreement with previous reports of Taxol| aster formation [1719]. In addition, the distance between the spindle poles at opposite ends of the cell was significantly shorter for the Taxol| cells relative to the control cells (4.0 +/- 0.4 ~m vs. 7.4 +/- 0.2 ~m). As the concentration of Taxol| increased, alterations in spindle formation became more pronounced. The chromosomes, which are normally aligned at the metaphase plate, were attached to microtubules which extended from the centriole like the spokes of a wheel. The normal bipolar configuration, with the two centrioles at opposite ends of the cell, was not observed in HeLa cells treated with micromolar concentrations of Taxol|

Some chromosomes were attached to astral microtubules which

also extended from the centriole.

311 At Taxol |

concentrations above 10 nM, an increase in the mass of

microtubule polymer was detected.

Maximal enhancement of microtubule

polymer levels was achieved at 330 nM Taxol | polymer mass.

In addition, at high Taxol |

with a 500% increase in

concentrations, microtubule

bundles were observed. Some loose bundles were detected in a few cells incubated with 33 nM Taxol| however, 1 pM Taxol| induced formation of massive microtubule bundles.

The effects of Taxol|

on HeLa cells at

concentrations above 10 nM, such as induction of microtubule bundles and increased polymer mass, are not surprising and have been previously reported for a number of cell lines. The most notable effects of Taxol | reported by Jordan et al. are those that occur at low drug concentrations [16]. In addition to investigating the effects of nanomolar levels of Taxol | on HeLa cells, the effects on microtubule dynamics in vitro were examined. Video-enhanced differential interference c o n t r a s t microscopy was employed to assess the dynamic instability of Taxol| microtubules assembled from pure bovine brain tubulin. In the presence of 0.1 and 0.5 ~M Taxol| both the microtubule growing and shortening rates were affected. These rates were inhibited by approximately 50% with 0.1 ~M Taxol (ii). In addition, the exchange of tubulin subunits at microtubule ends was inhibited substantially at 0.1 llM. This exchange process is defined as microtubule dynamicity and is defined as the number of tubulin dimers exchanged per second. Microtubule dynamicity was inhibited by 70 % with 0.5 ~tM Taxol| Taxol (i!) also influenced the length of time that a microtubule spent in an attenuated state.

The attenuated state defines that period in which a

microtubule is neither growing nor shortening, i.e. a static state. It is important to note that these concentrations of Taxol | 0.1 and 0.5 ~M, did not induce any detectable increases in microtubule polymer mass. Suppression of microtubule dynamics by antimitotic agents is an attractive mechanism for the biochemist/medicinal chemist. Although the primary effect of these compounds in vitro is thermodynamic (shifting of the monomer-polymer equilibrium), the in vivo effect which ultimately may prevent mitosis appears kinetic in nature.

Through subtle alterations in

the rates of monomer gain and loss at the microtubule ends, chromosomal segregation is disrupted.

312 7.3. THE BASIS F O R THE THERAPEUTIC EFFECTS OF TAXOL | One of the p r i m a r y goals of this review is to explore how the biochemical effects of Taxol | eventual cellular consequences. Taxol |

on tubulin and microtubules relate to its Although the primary cellular target of

appears to be t u b u l i n and microtubules, the in vivo steps

subsequent to Taxol |

binding are ill-defined.

In addition, Bhalla and co-

workers have d e m o n s t r a t e d t h a t the ul t i m at e consequence of Taxol | t r e a t m e n t in a leukemic cell line is apoptosis [35]. directed, energy-dependent process of cell death.

Apoptosis is a geneOne h a l l m a r k of this

process is fragmentation of cellular DNA into multiples of 200-base pair units by a Ca 2+ and M g 2 + - d e p e n d e n t endonuclease. characteristic

morphological

changes

are

also

A n u m b e r of

observed,

including

chromatin condensation, nuclear disintegration, and the formation of apoptotic cell bodies [36-39]. A number of potent and very effective chemotherapeutic drugs such as cytosine arabinoside, etoposide, cisplatin and doxorubicin have been shown to induce leukemic cell death by this apoptotic pathway.

In addition,

microtubule-selective agents such as colchicine and vinblastine have also been shown to induce apoptotic cell death in leukemic cells. Notably, the primary cellular targets of these drugs can vary, yet the same cellular markers of apoptosis are observed in leukemic cells t reat ed with these drugs.

This suggests t h a t interaction with the pri m ary target perturbs

cellular function in such a m a n n e r t hat a common signaling mechanism initiates cell death. The influence of antimitotic agents on microtubule dynamics described above attempts to rationalize how cells are sensitive to a diverse group of molecules. and Taxol |

Despite their seemingly opposite modes of action, vinblastine share a n u m b e r of common effects on microtubules, as

demonstrated by the work of Jordan and Wilson [15-21, 32-34]. However, their findings provide no clues about the selectivity of Taxol |

against a

select group of tumor types. Why does Taxol |

exhibit selective cytotoxicity toward some cancer

cells? The available data show that Taxol | has a narrow therapeutic index with significant efficacy against ovarian and breast tumors. which will ultimately define the selectivity of Taxol |

The factors

may be numerous.

313 Since all eukaryotic cells have intact tubulin]microtubule systems, the sensitivity of specific cell types to Taxol| must be influenced by factors which may be distinct from the tubulin/microtubule system. Table 1. Observed Effects of Taxol |

Biochemical Effects of Taxol|

References

Shifts monomer-polymer equilibrium toward polymeric state (affects thermodynamics of tubulin polymerization)

24-26

Affects the kinetics of monomer addition and loss at microtubule ends (microtubule dynamics)

15-21

Alters cofactor requirements (GTP, Mg 2+, etc. ) for tubulin polymerization

9, 31

Stabilizes microtubule against depolymerization induced by Ca 2+, dilution, or low temperatures

24-28

Cellular Effects of Taxol| Induction of microtubule bundling in leukemic cell line

70-75

Induction of mitotic arrest at the G2/M interface

24, 70

Induction of apoptosis in Taxol|

leukemic cells

Expression of multidrug transporter in resistant cells T axol| mimics the effects of LPS in murine macrophages

35 80-84 103-108

The tubulin-microtubule system is carefully regulated by a variety of physiological modulators, and it is likely that these endogenous regulators

314 will exert a significant influence on the effects of intracellular Taxol | Moreover, because the presence and nature of many modulating agents varies according to cell type and stage of development, these factors may contribute to the selectivity of Taxol (!i) in cellular targeting. The following sections will describe the effects of Taxol (ii) on the tubulin/microtubule system that have been identified at the biochemical and cellular levels. 7.4. C E L L U L A R

TUBULIN

CONCENTRATIONS

AND

ISOTYPE

COMPOSITION INFLUENCE TAXOL | SENSITIVITY The microtubules which form the mitotic spindle and the cytoskeleton within cells vary in their stability, ease of formation, and dynamic properties. The constant flux between soluble tubulin monomers and polymer is a fundamental property of microtubules which dictates the functional activities and diversity of microtubules. Cabral et al. have demonstrated that the levels of polymerized tubulin within cells correlates with drug resistance to antimitotic agents [40]. The antimitotic agents discussed thus far have all been shown to interact with microtubule ends and influence microtubule dynamics. Subtle changes in microtubule stability ( a d j u s t m e n t s in the monomer/polymer equilibrium) can influence both sensitivity and resistance to antimitotic agents. In an attempt to elucidate a novel mechanism of drug resistance (see section 7.7) the levels of polymerized tubulin in different drug-sensitive and drug-resistant cells were measured. In these experiments, Cabral et al. found t h a t CHO cells which were resistant to Taxol | but not colchicine or vinblastine, contained lower levels of polymerized tubulin ( - 2 0 - 3 0 % ) w h e n compared to the wild-type cells (-40%). These researchers suggested that the addition of Taxol | to the Taxol(ii)-resistant cells would enhance the cellular level of polymerized tubulin to near the wild-type level of polymer, but the increase in polymer mass would not be sufficient to affect viability. Conversely, CHO cells which were r e s i s t a n t to microtubule-depolymerizing agents, such as colchicine or vinblastine,

contained higher levels of polymerized tubulin

(-50%). In addition to explaining a novel mechanism of drug resistance, this hypothesis presented by Cabral and coworkers may provide a simple

315 indicator of cell sensitivity to Taxol | [40]. A cell with normally high levels of polymerized tubulin may be more sensitive to Taxol | than a cell with a lower level of polymer.

Investigation of cellular polymer levels in a wide

variety of cell types could be very useful in determining sensitivity to Taxol| In most eukaryotes, but particularly higher vertebrates, both r and [3tubulin exhibit considerable polymorphism. Both subunits are encoded by small multigene families and both are subject to post-translational modifications, including the addition and subsequent removal of the Cterminal tyrosine of a-tubulin [41,42] the acetylation of r [43-45], the polyglutamylation of a- and ~-tubulin [46,47], and the phosphorylation of ~-tubulin [48-51]. Additionally, the expression of different tubulin genes is differentially regulated during development and is in part either tissueor cell specific [52-54]. The various gene products are referred to as isotypes; the v a r i a n t s arising by post-translational modification are referred to as isoforms. A fundamental, unresolved issue is the extent to which the structural and functional characteristics of different microtubule populations are determined by differences in the primary sequence of tubulin isotypes. The evolutionary conservation across species of distinct isotypes, as well as the differential expression of different members of tubulin gene families, indicates that at least some isotypes may be functionally specialized with distinct biochemical functions in vivo. This proposal is known as "the multitubulin hypothesis" [55-57]. A closely related issue is the role of posttranslational modification in the function of tubulin variants. The size of the tubulin gene families can range from one to two a- and ~- tubulin genes for fungi to five to seven a- and ~-tubulin genes for higher vertebrates [58]. Mammalian brain is the most frequently used source of tubulin for structural and kinetic studies. At least five r and five ~-tubulin isotypes are expressed in the adult mammalian brain, which suggests that as few as five and as many as 25 structurally distinct a,~dimers may exist in the brain [56]. The post-translational modifications of these isotypes increase this number further. The precise functional roles of isotype/isoform species is not presently dear. Recent studies by Luduena and coworkers suggest that ~-tubulin isotype composition has an effect on the in vitro assembly of brain tubulin

316 [59]. Both the maximal rate and the extent of polymerization increases when bovine brain tubulin depleted of class III ~-tubulin by immunoaffinity chromatography is stimulated to assemble by either MAP2 or tau. Similarly, whereas very little is known about the role of tubulin posttranslational modification, recent studies by Frankfurter and co-workers indicate that the charge heterogeneity of rat brain tubulin affects in vitro MAP-stimulated assembly [60]. They have d e m o n s t r a t e d t h a t polyglutamylation of rat brain class III ~-tubulin increases with development [61] and, more recently, that the critical concentrations of tubulin required for assembly for postnatal day 10 and newborn MAP-free tubulin were 2-fold and 3-fold higher, respectively, than for adult tubulin. Moreover, the MAPstimulated assembly of adult tubulin exhibited biphasic kinetics (indicative of at least two tubulin populations), whereas postnatal day 10 and newborn tubulin assembled with monophasic kinetics [61]. With the sole exception of the acetylation of a-tubulin at lysine 40, the remaining known post-translational modifications of tubulin occur within the C-terminal isotype-defining domain, approximately the last 15 residues. What is compelling about this observation is that the isotype-defining Cterminal domain is believed to contain the binding sites for MAP2 and Ca 2+ [62]. Moreover, both polyglutamylation and phosphorylation of ~-tubulin occur in this region and are developmentally regulated [47]. Consequently, these p o s t - t r a n s l a t i o n a l modifications may significantly alter the interaction of tubulin with a number of ligands during neural development. For example, the ionic interactions which control the association of MAPs with tubulin are localized to this C-terminal domain [63]. The further acidification of this already negatively charged region might strengthen MAP binding and serve, in part, to increase microtubule stability. Present evidence is consistent with isotypes and isoforms as playing important roles in the kinetics and thermodynamics of tubulin assembly. The underlying chemotherapeutic utility of tubulin-specific agents may result from their ability to selectively bind to and stabilize a discrete subpopulation of tubulins. Class III ~-tubulin has been shown to be responsible for the slow phase of colchicine binding. Selective removal of class III ~-tubulin and its associated r from MAP-free tubulin alters the kinetics of colchicine binding from biphasic to monophasic [64]. In addition, the slow phase of colchicine binding was not observed for class

317 III-depleted tubulin. Microtubule depolymerization induced by colchicine selectively depolymerizes class III ~-tubulin first. Recently, Lu and Luduena reported that the removal of class III ~tubulin and its associated r enhanced Taxol| microtubule assembly [65]. The Class III-depleted tubulin assembled more rapidly and to a greater extent than unfractionated tubulin in the presence of Taxol | The critical concentration (Cc) of tubulin required for assembly in the presence of 10 ~M Taxol| was only 0.16 mg/ml for the Class IIIdepleted tubulin as compared to 0.4 mg/ml for the unfractionated tubulin. Their studies provide further evidence that tubulin isotype composition can greatly influence the kinetics of tubulin assembly. It is intriguing to hypothesize that a cell could modulate its sensitivity to antimitotic agents through alterations in its isotype composition. In our laboratory, we investigated the isotype-selectivity of Taxol| induced tubulin polymerization. Our goal was to assess the contribution of ~-tubulin isotype composition and charge heterogeneity on the kinetics of Taxol| microtubule assembly. Analysis of tubulin charge heterogeneity was achieved by high resolution isoelectric focusing (IEF). Microtubules, formed from MAP-free tubulin with varying concentrations of Taxol| were subjected to isoelectric focusing and immunostaining with isotype-specific antibodies to assess any variations in isotypes polymerized. These studies showed that Taxol| did not exhibit any selectivity under the experimental conditions employed (unpublished results). Consistent with this observation, photoaffinity labeled analogs of Taxol | have been employed to identify the binding site on tubulin. Using a 3H-labeled 3'-(p-azidobenzamido)taxol, Horwitz and co-workers recently localized the Taxol| binding site to the N-terminal 31 amino acids of ~tubulin [66]. The N-terminal region of the tubulins, unlike the C-terminal region, is much more conserved across isotypes. Consequently, Taxol | binding to this N-terminal domain would not be expected to show any isotype selectivity. 7.5. TAXOL| :INDUCES MICROTUBUI~ BUNDLING The cylindrical structure of microtubules results in a fairly rigid structure with limited conformational motility. In vivo, individual

318 microtubules, composed of tubulin, are decorated with microtubuleassociated proteins (MAPs). Much evidence suggests that MAPs extend from the surface of the microtubule and influence interactions between adjacent microtubules. Longitudinally-associated microtubule bundles have been observed in a number of cell types. The formation of any unusual or aberrant microtubule structure which may suggest a plausible mechanism of action of antimitotic agents is of interest. The interplay of cellular proteins and the mitotic machinery may provide the most compelling clues about the cellular mechanisms of these agents. Taxol| has been shown to influence the number of protofilaments within a microtubule structure in vivo [67]. Whereas the majority of animal and p l a n t cells construct m i c r o t u b u l e s c o n t a i n i n g 13 protofilaments, developing wings of Drosophilia construct 15 protofilament microtubules [68]. Taxol| treatment of cultured wings induced formation of microtubules containing 12 protofilaments at microtubule-nucleating sites. Dye et al. have d e m o n s t r a t e d by video-enhanced differential interference contrast microscopy t h a t Taxol| microtubules, assembled in vitro from pure tubulin, became flexible and appeared wavy [69]. Notably, the addition of MAP2 or tau, two major MAPs, reversed this Taxol| flexibility. The addition of these MAPs did not cause Taxol| to dissociate from the microtubule, since flexibility was again observed when the MAPs were released from the microtubules by high salt concentrations. This alteration in the flexibility of microtubules is likely to result from slippage between adjacent protofilaments in the cylindrical microtubule structure. A decrease in the strength of the lateral or circumferential interactions induced by Taxol| would allow bending of the microtubule structure. Reversal of this phenomenon by MAPs may result from the ability of these proteins to bridge adjacent protofilaments and restore the strength of the lateral protofilament interactions. This observation of Taxol| microtubule flexibility and its reversal by MAP2 and tau may have relevant consequences in vivo. Although MAPs are always associated with microtubules in vivo, their distribution and levels of expression vary in different cell types. Thus, the microtubules in Taxol| cells may have enhanced flexibility, which

319 would affect their ability to perform crucial cellular functions requiring rigid microtubule structures. In fact, alterations in microtubule flexibility may explain the ability of Taxol| to induce microtubule bundling i n vivo. The formation of microtubule bundles in Taxol| cells has been observed. Microtubule bundles can be described as abnormal microtubule arrays composed of multiple microtubules which associate with each other longitudinally. Rowinsky and coworkers have d e m o n s t r a t e d t h a t the stability of these Taxol| microtubule bundles correlates well with cell sensitivity to Taxol| [70-75]. Taxol | t r e a t m e n t of cultured leukemic cells has been shown to induce mitotic arrest at the G2/M interface of the cell cycle. However, the formation of microtubule bundles in these cell lines was observed t h r o u g h o u t the cell cycle. Taxol| t r e a t m e n t induced microtubule bundling in sensitive leukemic cell lines such as HL-60 and LC8A, and also in the relatively resistant cell lines K562 and Daudi [70]. Microtubule bundling in the resistant cell lines was reversible both in the presence and absence of Taxol| and these cells accumulated in G2/M. Conversely, the microtubule bundles formed in the sensitive cell lines appeared to be irreversible and persisted in the presence and absence of Taxol| Thus, the stability, not simply the formation, of the microtubule bundles correlates with the cytotoxicity of Taxol | Another observed microtubule change in these T a x o l | leukemic cell lines was the formation of multiple aster-like aggregates of short microtubules [71-75]. Asters, also composed of microtubules, radiate from the centrioles in a star-like conformation during mitosis. The role of astral microtubules in mitosis is not well understood. The formation of multiple asters and microtubule bundles were independent events and did not occur simultaneously. In addition, the formation of these multiple "asters" did not correlate well with sensitivity to Taxol| Subsequently, these researchers demonstrated the cell cycle phase of the Taxol| formation of multiple asters and microtubule bundles [71]. The formation of Taxol| multiple asters occurred during mitosis, whereas cells containing microtubule bundles were in G0/G1, S, and G2 phases of the cell cycle. Both a Taxol| and a Taxol| resistant cell line displayed Taxol| asters only during mitosis. In addition, there were no notable variations in the cell cycle positions for microtubule bundle formation in the Taxol|

vs. the Taxol |

320 resistant cell lines. Hence, the sensitivity of a cell line to Taxol | does not correlate with microtubule bundle formation during a particular phase of the cell cycle or with the ability of the Taxol| cell to synthesize DNA (S-phase specific). The majority of studies have demonstrated that the cytotoxicity of the V i n c a alkaloids (such as vinblastine) and other tubulin-specific agents is due to their effect on microtubules, specifically the mitotic spindle. However, recent evidence suggests that the cytotoxicity of these compounds may result from effects on cells during interphase [72, 73]. Roberts et al. compared the effect of Taxol | on S-phase activity (DNA synthesis) in both a Taxol| (HL60) and a T a x o l | leukemic cell line (K562) [74]. Although the labeling of DNA with [3H]thymidine was scarcely affected in these two cell lines (a measure of Sphase activity), the formation of a high percentage of polyploid cells in the Taxol| cell line, K562, was observed. Following 24 hour t r e a t m e n t with Taxol | 66% of the Taxol| K562 cells contained multiple numbers of chromosomes (polyploidization), whereas only 8% of the Taxol| HL60 cells were polyploid. The induction of polyploidism in Taxol| cell lines relative to Taxol| cell lines suggests t h a t this may be a useful indicator of drug sensitivity. Resistance of a cell line to Taxol | may be predicted based on its ability to induce such a distinct alteration in chromosome numbers. Thus, Rowinsky and coworkers have demonstrated that the formation of persistent microtubule bundles correlates with Taxol | sensitivity in leukemic cell lines, although the formation of these persistent structures is not cell-cycle specific. In addition to direct effects on microtubules, Taxol | treatment induced polyploidism to a great extent only in Taxol| leukemic cell lines. These observations provide researchers with clear cellular markers to assess sensitivity to Taxol | [70-75]. The formation of stable microtubule bundles in Taxol| cells suggests that these structures play a crucial role in mediating the cytotoxic activity of Taxol | Therefore, the biochemical mechanisms of bundle formation and the consequences of this event have been investigated. Turner and Margolis have examined the bundling of Taxol| microtubules in vitro [76]. Their study examined the effect of ATP on bundle formation and also identified a protein factor which induced bundle

321 formation in vitro. The protein factor which was required for in vitro bundling did not copurify with tubulin through cycles of assembly and disassembly, which indicates that it is not a microtubule-associated protein (MAP). However, Cowan and coworkers have reported t h a t microtubule bundling into parallel arrays is induced by MAP2 and tau, both of which are MAPs [77]. Crosslinking of adjacent microtubules is achieved by a short C-terminal sequence present in both MAP2 and tau. This a-helical domain is distinct from the microtubule-binding site which serves to nucleate microtubule assembly. Construction of a n u m b e r of m u t a n t MAP2 proteins has allowed the domain which influences bundle formation to be identifed [78]. The microtubule bundles formed in the presence of Taxol| which were observed by T u r n e r and Margolis were sensitive to high c o n c e n t r a t i o n s of ATP, which s u g g e s t s a p h o s p h o r y l a t i o n - l i n k e d mechanism. In addition, elevated Ca 2+ concentrations (millimolar) relaxed Taxol| microtubule bundles and released s u b s t a n t i a l amounts of MAPs from these structures. Relaxation of Taxol| bundles by high concentrations of Ca 2+ and ATP implicates signal transduction pathways in the regulation of this drug-induced event. U m e y a m a and coworkers examined the dynamics of microtubules which were induced to bundle by MAP2C in vivo [79]. Both the incorporation of biotin-labeled exogenous tubulin into the microtubule ends and the turnover rate of microtubule bundles were investigated in this study. Incorporation of the biotin-labeled tubulin into preexisting microtubule ends was fairly rapid. However, the microtubule turnover rate, determined by photoactivation of caged fluorescein-labeled tubulin, was dramatically reduced. Normally there is a constant flux between the monomer and polymer pools of tubulin within cells. The abnormal microtubule bundling array induced by MAP2C in this study prevented this normally facile flux. The observation t h a t microtubule bundles have altered dynamic properties is not surprising. Although, in this case, the bundles were induced by MAP2C, cellular microtubule bundles formed in the presence of Taxol|

would be anticipated to exhibit similar losses in dynamics.

These

322 data lend further support to the hypothesis that suppression of microtubule dynamics by antimitotic agents is the mechanism of cytotoxicity.

7.6. MECHANISMS OF RESISTANCE TO TAXOL| R e s i s t a n c e to Taxol| h a s s u b s t a n t i a l l y l i m i t e d its clinical development. Due to its hydrophobic nature, Taxol| induces overexpression of the m u l t i d r u g t r a n s p o r t e r , P-glycoprotein [80, 81]. Pglycoprotein is a membrane-spanning glycoprotein which is found in most normal cells in low amounts, but its gene (mdr) is amplified in drugresistant cells. Enhanced levels of this protein increase the cell's ability to remove a c c u m u l a t e d hydrophobic drugs. The compounds which are extruded by this ATP-driven pump are diverse and affect a n u m b e r of different i n t r a c e l l u l a r targets in addition to the tubulin/microtubule system, such as DNA topoisomerase II. Roy and Horwitz first identified a phosphoglycoprotein associated with Taxol | resistance in J774.2 cells, a murine macrophage-like cell line [82]. Drug resistance was induced by growing the cells in the presence of increasing concentrations of Taxol| Cross-resistance to microtubule-specific agents, such as colchicine and vinblastine as well as compounds such as doxorubicin and actinomycin D, was observed. Analysis of membrane proteins in the Taxol| and wild-type cells by SDS-PAGE revealed a prominent protein band at 135 kDa in the Taxol| cells which was scarcely detected in the wild-type cells. The presence of this protein band correlated well with drug resistance to Taxol| In addition, colchicine- and vinblastine-resistant J774.2 cells displayed prominent phosphoglycoprotein bands at 145 and 150 kDa, respectively. Thus, the phosphoglycoprotein of the Taxol| cell lines is similar, but not identical, to the classic P-glycoprotein expressed in vinblastine- and colchicine-resistant cell lines. The full-length cDNA of the m d r l gene encodes a 170 kDa P-glycoprotein. The detection of these smaller membrane phosphoproteins in drug-resistant cell lines suggests that a number of heterogeneous drug transporters exist. Greenberger and co-workers have examined the biosynthesis of the heterogeneous forms of glycoproteins which are expressed in drug resistant J774.2 cells [83, 84].

The heterogeneity in vinblastine- and

323 colchicine-resistant cells lines was attributed to variations in glycosylation which resulted in altered electrophoretic mobility. In these vinblastineand colchicine- resistant cells, a single precursor (125-kDa) was rapidly processed to two forms of 135- and 140kDa. However, in Taxol| J774.2 cells, two distinct precursor proteins were expressed [83]. This observation demonstrated that different glycoproteins which mediate drug resistance could be expressed in response to different drugs. In order to f u r t h e r investigate this heterogeneity, the forms of P-glycoprotein expressed in independent cell lines were examined [84]. The results of this study indicated t h a t expression of the precursor forms of the d r u g - r e s i s t a n c e - a s s o c i a t e d proteins was not drug specific when examined over a range of cell types. A number of approaches have been employed in order to overcome Pglycoprotein-mediated Taxol| resistance. The sensitivity of resistant cells to Taxol| was restored when Taxol | was administered in conjunction with agents such as the cyclosporine derivative SDZ PSC 833 and the cyclopeptolide SDZ 280-446 [85]. Combination drug t h e r a p y may be necessary to overcome r e s i s t a n c e to Taxol | C u r r e n t l y Taxol | is administered with cremophor-EL, which enhances solubility and also drug accumulation intracellularly [86]. Recently, Taxol| has been administered in drug carriers such as liposomes. The interactions of Taxol | with lipids have been studied as well. The investigation of the interactions of Taxol | with dipalmitoyl phosphatidylcholine (DPPC) liposomes by a number of physical methods demonstrated t h a t the drug can partition into the membrane bilayer and perturb the m e m b r a n e [87]. Membrane fluidity and the lipid order p a r a m e t e r were affected. These findings are clinically relevant, since cellular uptake is a prerequisite for effective drug/target interactions. Bhalla and coworkers had previously demonstrated that t r e a t m e n t of leukemic cell lines with clinically relevant levels of Taxol| induced the c h a r a c t e r i s t i c morphological changes associated with apoptosis or programmed cell death [35]. In that earlier study, Bhalla and coworkers had also d e m o n s t r a t e d t h a t Taxol| t r e a t m e n t altered the levels of expression of two oncogene products, bcl-2 and c-myc. Bcl-2 has been d e m o n s t r a t e d to block apoptotic cell d e a t h t h r o u g h an ill-defined a n t i o x i d a n t p a t h w a y [88] and cellular levels of this mitochondrial

324 membrane protein appear to correlate with resistance to antitumor agents. Recently, Miyashita and Reed demonstrated that elevated cellular levels of the bcl-2 oncoprotein could block apoptosis induced by chemotherapeutic agents in a h u m a n leukemia cell line [89]. In addition, leukemic cells which are arrested at the G2/M transition of the cell cycle, such as those treated with Taxol| typically undergo apoptosis. In an attempt to further elucidate cellular mechanisms of resistance to Taxol| as well as other antitumor agents, Bhalla and coworkers have characterized a Taxol| h u m a n myeloid leukemia cell line, HL-60 [90]. Given previous results, these researchers examined the levels of expression of the multidrug transporter, P-glycoprotein, as well as the levels of bcl-2. These Taxol| cells overexpressed P-glycoprotein and, as a result, exhibited cross-resistance to other drugs including vincristine and doxorubicin. In addition, Taxol | t r e a t m e n t did not induce microtubule bundle formation in these resistant cells and the cells did not undergo apoptosis. Examination of the cellular levels of bcl-2 by Western blot analysis revealed t h a t the Taxol| cells did not have significantly elevated levels of bcl-2 relative to the Taxol| cells. This suggests t h a t Taxol | resistance in this cell line is not due to overexpression of bcl-2. The absence of microtubule bundles in the Taxol| resistant HL-60 cells is consistent with the observations of Rowinsky and coworkers, who have d e m o n s t r a t e d t h a t the presence of p e r s i s t e n t microtubule bundles correlates with sensitivity to Taxol| [70-75]. Although the levels of bcl-2 in the Taxol| leukemic cell line were not elevated, the role of bcl-2 in cellular resistance to antitumor agents is of considerable interest. Most recently, Willingham and Bhalla used an anti-bcl-2 monoclonal antibody and fluorescence immunocytochemistry to localize the bcl-2 protein during mitosis in h u m a n carcinoma cell lines, KB and OVCAR-3 [91]. In both these cell lines, interphase cells showed no detectable expression of bcl-2. However, cells undergoing mitosis displayed localized bcl-2 protein around condensed chromosomes. This pattern of bcl2 localization was observed in prophase, metaphase and anaphase in the two cell lines studied. Cells in telophase no longer contained detectable bcl2 protein. Taxol | treatment of these cell lines did not alter the distribution of bcl2 around the condensed chromosomes during mitosis, but mitotic arrest

325 was observed, and with continued Taxol (!i) treatment the cells showed the characteristic morphological changes associated with apoptosis. In additon, bcl-2 localization was lost. Previously, Taxol | had been demonstrated to induce apoptosis in leukemic cell lines [35]. These results of apoptosis induced by Taxol | in human carcinoma cell lines support a common mechanistic outcome of Taxol |

treatment in mammalian cells.

The relevance of these bcl-2 localization studies during mitosis is considerable. The observation that bcl-2 is expressed and associates with chromosomes at the initiation of mitosis, and that the protein disappears in telophase, suggests a protective role during the time the chromosomes are accessible to the cytoplasm.

Bcl-2 may protect the chromosomes from

degradation by the "apoptotic" nuclease at internucleosomal sites. If this were the case, overepression of bcl-2 could prevent apoptosis by preventing DNA damage. Since treatment with Taxol | induces mitotic arrest in these cells, the length of time the chromosomes are exposed in the cytoplasm is altered and the protective action of bcl-2 may be affected.

The continued

investigation of the role of bcl-2 in mitosis and apoptosis may provide important insights into the mechanism of antimitotic agents such as Taxol (ii). The levels of bcl-2 during mitosis in different cell lines could help address the issue of Taxol | selectivity against a particular tumor cell line. A novel mechanism of resistance to microtubule-specific agents has been observed by Cabral and co-workers [40, 92-98]. Random mutagenesis by ultraviolet irradiation was employed to generate drug-resistant CHO cell lines. Early investigation of these drug-resistant mutants produced by random mutagenesis demonstrated that resistance was not the result of amplification of P-glycoprotein or defects in drug accumulation. These drug-resistant CHO cell mutants were found to have altered a - a n d 13tubulins, as determined by two-dimensional electrophoresis [98]. However, the mutant tubulin subunits did not have altered drug-binding properties, which would be the simple hypothesis to explain the observed drug resistance.

Nonetheless, a direct effect on microtubules is supported by

evidence that cells resistant to a destabilizing drug such as colcemid are cross-resistant to other destabilizing agents even though their binding sites on tubulin are distinct.

In addition, cells r e s i s t a n t to microtubule-

destabilizing agents are hypersensitive to Taxol | agent.

a microtubule stabilizing

326 Random mutagenesis of CHO cells by ultraviolet radiation produced a n u m b e r of m u t a n t s which were r e s i s t a n t to Taxol | a microtubulestabilizing agent, and colcemid, a microtubule-destabilizing agent. In addition, a m u t a n t which required Taxol| for survival was also identified [96, 97]. In the absence of Taxol| the CHO cell m u t a n t which required Taxol| for survival initially accumulated in G 2 ~ , and did not undergo cell division (cytokinesis), but f u r t h e r increased its DNA content (became polyploid). This indicates t h a t the mutation does not halt progression through the cell cycle, but it prevents cytokinesis, the ultimate splitting of the parent cell into two daughter cells. The Taxol| m u t a n t did not form a functional mitotic spindle apparatus in the absence of Taxol | as d e t e r m i n e d by i m m u n o f l u o r e s c e n c e studies, a l t h o u g h non-spindle cytoplasmic microtubules were formed. The spindle microtubules which did form in the absence of Taxol| were shorter, fewer in number, and were kinetochore microtubules only. The mitotic spindle contains two forms of microtubules: the kinetochore microtubules, which radiate from the centrioles and form the contact site of microtubule a t t a c h m e n t to the chromosome at the centromere, and interpolar microtubules, which interdigitate between the chromosomes which are aligned at the metaphase plate [99]. In a d d i t i o n , i n t e r p o l a r microtubules known as asters extend from the centrioles in a star-like conformation. The kinetochore microtubules are more stable t h a n the interpolar microtubules and they are believed to be responsible for the chromosomal segregation which begins during anaphase [100, 101]. Initial studies of the Taxol| CHO cell m u t a n t suggested that the cells contained lower intracellular concentrations of tubulin, which allowed cytoplasmic microtubules to form, b u t not spindle microtubules. Addition of Taxol| would shift the monomer-polymer equilibrium t o w a r d the polymerized state and reduce the critical concentration of tubulin required for microtubule assembly. As a result, spindle microtubules would be able to form. However, quantitation of intracellular tubulin did not reveal significant alterations in the levels of total tubulin in these Taxol| cells. In order to further probe the nature of this resistance to antimitotic drugs, Minotti et al. have examined the levels of polymerized tubulin in drug-resistant CHO cells [40].

Since subtle changes in microtubule

327 stability (adjustments in the monomer-polymer equilibrium) are believed to be responsible for resistance to antimitotic agents in these mutants, the levels of polymerized tubulin in these cells may correlate with drug resistance. As anticipated, Taxol| mutants contained lower amounts of polymerized tubulin (-20-30%) relative to wild-type CHO cells (-40%). Thus, addition of Taxol| to these resistant cell lines would enhance the amount of polymerized tubulin, but the increase in polymer would not be sufficient to affect cell viability. Conversely, m u t a n t s r e s i s t a n t to microtubuledestabilizing agents, such as colcemid and vinblastine, contained elevated levels of polymer (-50%) relative to the wild-type cells. These effects on the levels of polymerized tubulin confirm that subtle alterations in the microtubule assembly properties of CHO m u t a n t s can confer drug resistance. This r e p r e s e n t s a common m e c h a n i s m of resistance to both microtubule-stabilizing and destabilizing agents. This mechanism is distinct from drug resistance mediated by overexpression of P-glycoprotein, a multidrug transporter. In addition, the sensitivity of a particular cell line to Taxol| or other antimitotic agents may be related to the level of polymerized tubulin in those cells. The levels of polymerized tubulin in different cell lines or tissues determined in a number of studies are highly variable. The selectivity of Taxol| against specific tumor types may be influenced by these variations in levels of polymerized tubulin. The need for more soluble Taxol| analogs has resulted in the development of Taxotere | a semi-synthetic analog with enhanced solubility and equal or better activity. Diaz and Andreu have recently compared the assembly of purified tubulin to microtubules induced by Taxol| T a x o t e r e | [102]. Their data confirm t h a t Taxol| and Taxotere | compete for the same binding site on microtubules. Notably, the critical concentration of tubulin required for microtubule formation was 2.1 +/- 0.1 times smaller with Taxotere | than with Taxol| 7.7. ALTERNATIVE AND SECONDARY E F F E C T S OF TAXOL| Although Taxol| interacts primarily with the tubulin]microtubule system, a number of in vivo Taxol| effects that appear independent of the tubulin]microtubule system have been reported. While investigating the

328 effects of microtubule targeting agents on tumor necrosis factor (TNF-a) receptors, Ding and co-workers observed that Taxol | profoundly affected murine macrophages [103]. Taxol | was found to induce TNF-(z release and decreased expression of the TNF receptor.

These effects mediated by

Taxol |

were identical to those elicited by bacterial lipopolysaccharide

(LPS).

These data suggest that the actions of LPS on macrophages are

mediated by an i n t r a c e l l u l a r t a r g e t also affected by Taxol |

The

intracellular target is suggested to be a microtubule-associated protein, rather than tubulin, and the presence of Taxol | function of an unidentified MAP.

may disrupt the normal

-In a subsequent study by Manthey and coworkers, the ability of Taxol | to induce gene expression and to generate a LPS-like signal in murine macrophages was examined [104]. Taxol | was found to increase the steady-state levels of LPS-inducible genes and to induce the tyrosine phosphorylation of several proteins with molecular weights of 41-45 kDa. Further elucidation of a common signaling pathway mediated by LPS and Taxol | may provide crucial insight into the cellular mechanisms by which Taxol | influences tumor growth. Continued efforts of Manthey and coworkers have demonstrated that the LPS-like activities of Taxol | in murine macrophages are distinct from the effects of Taxol | on microtubule structure and stability [105]. Two lipopolysaccharide antagonists, RsDPLA and SDZ 880.431, inhibited Taxol| TNF-r release, gene a c t i v a t i o n and tyrosine phosphorylation. However, these drugs were unable to inhibit Taxol | induced microtubule bundling in RAW 264.7 macrophages. Taxotere | a semisynthetic analog of Taxol | (see Figure 3), did not induce TNF-a release or gene expression but was more effective at inducing microtubule bundle formation. Microtubule bundling was observed in RAW 264.7 macrophages in the presence of as little as 75 nM Taxotere | whereas 300 nM Taxol | was required to achieve the same level of bundle formation. It is the view of Manthey and coworkers t h a t Taxol |

activates

macrophages through an as yet unidentified protein which also mediates LPS-induced signaling. Identification of the components of such a pathway will provide insight into the cellular mechanism of Taxol|

antitumor

activity. The antitumor activity of LPS has been documented and this activity is directly linked to LPS's ability to induce the release of TNF-a and

329 other cytokines [106].

In addition, LPS serves as a signal to activate

macrophage nitric oxide synthase. However, the observation t h a t the Taxol| analog, Taxotere | is unable to activate gene expression or TNF-a release suggests t h a t these secondary cellular effects of Taxol| on murine macrophages are not required for the therapeutic activity of Taxol| derivatives. Taxotere | with its e n h a n c e d w a t e r solubility and microtubule-stabilization activity, is currently in Phase II clinical trials and is highly effective against breast cancer and other solid tumors [102]. Continued efforts by Manthey and co-workers to elucidate the role of Taxol| LPS-like signaling have demonstrated that this antimitotic drug provides a second signal for murine macrophage tumoricidal activity [107]. The combination of LPS or Taxol| and IFN-y induced macrophages to lyse tumor cells. This synergism required a functional LPS signaling pathway, as d e m o n s t r a t e d by using both LPS-responsive and LPShyporesponsive macrophage cell lines. In addition, the combination of LPS or Taxol| and IFN-y induced expression of nitric oxide synthase and, consequently, increased nitric oxide secretion. Cellular levels of nitric oxide correlated with tumor cell killing and addition of the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine, inhibited t u m o r cell killing. This work demonstrates that Taxol | has the potential to activate host a n t i t u m o r mechanisms which appear distinct from the effects of Taxol| on tubulin and microtubules. Ding and co-workers, who initially reported the shared activities of LPS and Taxol| have more recently examined the ability of Taxol| and LPS to induce the tyrosine phosphorylation of a microtubule-associated protein kinase, MAPK [108]. Phosphorylation is a common intracellular signaling event which has been shown to modulate the interaction of tubulin with MAPs. Tyrosine phosphorylation of MAPK, induced by Taxol | or LPS in murine macrophages, also triggered phosphorylation of an unidentified protein of approximately 86 kDa. MAPK, known as mitogenactivated protein kinase as well as a number of different names, is a family of s e r i n e / t h r e o n i n e k i n a s e s r e g u l a t e d by tyrosine a n d t h r e o n i n e phosphorylation. Induction of MAPK phosphorylation induced by LPS or Taxol|

was extremely rapid with near maximal levels of phosphate

incorporation within 1 minute.

Despite these recent findings, it is still

unknown whether these Taxol|

effects are all secondary to the interaction

330 of Taxol |

with microtubules. LPS has also been shown to bind to

microtubules. The possible links between microtubule binding and the LPS-like activities of Taxol |

seem to implicate microtubule-associated proteins in

the cellular mechanism of this antimitotic agent.

MAPK is a member of a

complex kinase cascade and the identification of u p s t r e a m activators of this cascade may provide scientists with clues as to the possible links between these two Taxol | activities.

It is important to note that a number

of proto-oncogene products including mos, fyn, and lyn are microtubuleassociated and some are tyrosine kinases [109, 110] which could act as upstream activators of MAPK.

Tyrosine phosphorylation of MAPK by a

microtubule-associated proto-oncogene product could be induced by a common Taxol|

pathway.

7.8. C O N C L U S I O N S

This review seeks to provide i n s i g h t s into the f u n d a m e n t a l mechanisms underlying the therapeutic utility of Taxol | by describing several critical biochemical and cellular effects of this antimitotic agent. U n d e r s t a n d i n g the interactions of Taxol |

with its p r i m a r y target, the

tubulin/microtubule system, at the biochemical level is vital to our ultimate understanding of the cellular consequences of Taxol | The ability of Taxol |

treatment.

and other antimitotic agents to suppress

microtubule dynamics both in vitro and in vivo provides the medicinal chemist with mechanistic insights as to how mitosis may be blocked. Ultimately, the roles of the numerous microtubule-associated proteins (MAPs) may provide some of the most compelling mechanistic clues as they may link the biochemical effects of Taxol |

on microtubules with the

cellular outcome of Taxol | treatment, which is apoptosis. Through continued efforts, f u n d a m e n t a l knowledge r e g a r d i n g the molecular mechanisms and the target sites of Taxol | cellular components will be acquired. fostering more effective Taxol |

interaction with

This knowledge can be applied to

use in cancer t r e a t m e n t , to predicting

additional sites for chemotherapeutic application, and to the development of additional therapeutic protocols.

331

o

2. 3. 4. ,

,

7. .

.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Dustin, P. Microtubules, 2nd Ed. Berlin: Springer-Verlag, 1984. Raft, E.C.J. Cell Biol. 1984 99, 1. Weisenberg, R.C. Science 1972,177, 1104. Weingarten, M.D.; Lockwood, A.H.; Hwo, S.Y. Proc. Natl. Acad. Sci. USA 1975, 72, 7858. Diaz-Nido, J.; Hernandez, M.A.; Avila, J. In: Microtubule Proteins. Boca Raton: CRC Press, 1990, p. 193. Williams, R.C.; Lee, J.C. Methods in Enzymology 1982, 85, 376. Wilson, L.; Jordan, M.A. In Microtubules; Hyams, J.S.; Lloyd, C.W. Eds.; New York: Wiley-Liss, 1994, p. 59. Hamel, E. In Microtubule Proteins; Boca Raton: CRC Press, 1990, p. 89. Carlier, M.-F.; Pantaloni, D. Biochemistry 1981, 20, 1918. Gilman, A.G. Ann. Rev. Biochem. 1987, 56, 615. Correia, J.J.; Williams, R.C. Ann. Rev. Biophys. Bioeng. 1983, 12,211. Gelfand, V.I.; Bershadsky, A.D. Ann. Rev. Cell Biol. 1991, 7, 93. Bayley, P.M.; Sharma, K.K.; Martin, S.R. In Microtubules; Hyams, J.S.; Lloyd, C.W., Eds.. New York: Wiley-Liss, 1994, p. 111. Mitchison, T.; Kirschner, M. Nature 1984, 312,347. Toso, R.J.; Jordan, M.A.; Farrell, K.W.; Matsumoto, B.; Wilson, L. Biochemistry 1993, 32, 1285. Jordan, M.A.; Toso, R.J.; Thrower, D.; Wilson, L. Proc. Natl. Acad. Sci. USA 1993, 90, 9552. Wendell, K.L.; Wilson, L.; Jordan, M.A.J. Cell Science 1993, 104, 261. Jordan, M.A.; Thrower, D.; Wilson, L. J. Cell Science 1992, 102,401. Jordan, M.A.; Thrower, D.; Wilson, L. Cancer Res. 1991, 51, 2212. Jordan, M.A.; Wilson, L. Biochemistry 1990, 29, 2730. Farrell, K.W.; Jordan, M.A.; Miller, H.P.; Wilson, L. J. Cell Science 1987, 184, 1035. Hastie, S.B. Pharmacol. Ther. 1991, 52, 377. Correia, J.J. Pharmacol. Ther. 1991, 52, 127. Schiff, P.B.; Font, J.; Horwitz, S.B. Nature 1979, 277, 665. Schiff, P.B.; Horwitz, S.B. Proc. Natl. Acad. Sci. USA 1980, 77, 1561. Schiff, P.B.; Horwitz, S.B. Biochemistry 1981, 20, 3247.

332 27. Manfredi, J.; Horwitz, S.B. Pharmacol. Ther. 1984, 25, 83. 28. Horwitz, S.B. TiPS 1992, 13, 134. 29. Timasheff, S.N.; Andreu, J.M.; Na, G.C. Pharmacol. Ther. 1991,52,191. 30. Howard, W.D.; Timasheff, S.N.J. Biol. Chem. 1988, 263, 1342. 31. Carlier, M-F.; Pantaloni, D. Biochemistry 1983, 22, 4814. 32. Wilson, L.; Miller, H.P.; Farrell, K.W.; Snyder, K.B.; Thompson, W.C.; Purich, D.L. Biochemistry 1985, 24, 5254. 33. Wilson, L.; Anderson, K.; Chin, D. In Cell Motility; Goldman, R.; Pollard, T.; Rosenbaum, J.L., Eds.; Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1976, p.1051. 34. Wilson, L.; Jordan, M.A.; Morse, A.; Margolis, R.L.J. Mol. Biol. 1982, 159, 129. 35. Bhalla, K.; Ibrado, A.M.; Tourkina, E.; Tang, C.; Mahoney, M.E.; Huang, Y. Leukemia 1993, 7, 563. 36. Martin, S.J.; Green, D.R.; Cotter, T.G. TiBS 1994, 19, 26. 37. Kamesaki, S.; Kamesaki, H.; Jorgensen, T.J.; Tanizawa, A.; Pommier, Y.; Cossman, J. Cancer Res. 1993, 53, 4251. 38. Gorczyca, W.; Bigman, K.; Mittelman, A.; Ahmed, T.; Gong, J.; Melamed, M.R.; Darzynkiewicz, Z. Leukemia 1993, 7, 659. 39. Hickman, J.A. Cancer Metast. Rev. 1992, 11, 121. 40. Minotti, A.M.; Barlow, S.B.; Cabral, F. J. Biol. Chem. 1991, 266, 3987. 41. Barra, H.S.; Arce, C.A.; Rodrigues, J.A.; Caputo, R. Biochem. Biophys. Res. Commun. 1974, 60, 1384. 42. Raybin, D.; Flavin, M. Biochem. Biophys. Res. Commun. 1975, 65, 1086. 43. L'Hernault, S.W.; Rosenbaum, J.L. Biochemistry 1985, 24,473. 44. Piperno, G.; Fuller, M. J Cell Biol. 1985, 101, 2085. 45. Black, M.M.; Keyser, P. J. Neurosci. 1986, 7, 1833. 46. Edde, B.; Rossier, J.; Le Caer, J-P.; Desbruyeres, E.; Gros, F.; Denoulet, P. Science 1990, 247, 83. 47. Alexander, J.E.; Hunt, D.F.; Lee, M.K.; Shabanowitz, J.; Michel, H.; Berlin, S.C.; Macdonald, T.L.; Sundberg, R.J.; Rebhun, L.R.; Frankfurter, A. Proc. Natl. Acad. Sci. USA 1991, 88, 4685. 48. Eipper, B. Proc. Natl. Acad. Sci. USA 1972, 69, 2283. 49. Luduena, R.F.; Zimmerman, H.; Little, M. FEBS Lett. 1988, 230, 142. 50. Gard, D.L.; Kirschner, M. J. Cell Biol. 1985, 100, 746.

333 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 4.

65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75.

Bond, J.F.; Robinson, G.C.; Farmer, S.R. Mol. Cell Biol. 1984, 4, 1313. Havercrott, J.C.; Cleveland, D.W.J. Cell Biol. 1984, 94, 1927. Lewis, S.A.; Cowan, N . J . J . Cell Biol. 1988, 106, 2023. Cleveland, D.W.J. Cell Biol. 1987, 104,381. Cleveland, D.W; Sullivan, K.F. Ann. Rev. Biochem. 1985, 54, 331. Sullivan, K.F. Ann. Rev. Cell Biol. 1988, 4, 687. Luduena, R.F. Mol. Biol. of the Cell 1993, 4, 445. Raft, E.C. In Microtubules; Hyams, J.S.; Lloyd, C.W., Eds.; New York: Wiley-Liss, Inc., 1994; p.85. Banerjee, A.; Roach, A.C.; Lopata, K.A.; Lopata, M.A.; Cleveland, D.W.; Luduena, R.F.J. Biol. Chem. 1988, 263, 3029. Lee, M.K.; Rebhun, L.I.; Frankfurter, A. Am. Soc. Cell Biol. 1990, abstract. Frankfurter, A.; Alexander, J.; Lee, M.K.; Hunt, D.F., 1990, unpublished results. Serrano, L.; Avila, J.; Maccioni, R.B. Biochemistry 1984, 23, 4675. Redeker, V.; Melki, R.; Prome, D.; Le Caer, J.P.; Rossier, J. FEBS Lett. 1992,313, 185. Banerjee, A.; Luduena, R.F.J. Biol. Chem. 1991, 266, 1689. Lu, Q.; Luduena, R.F. Cell Struct. and Funct. 1993, 18, 173. Rao, S.; Krauss, N.E.; Heerding, J.M.; Swindell, C.S.; Ringel, I.; Orr, G.A.; Horwitz, S.B.J. Biol. Chem. 1994, 269, 3132. Mogensen, M.M.; Tucker, J.B.J. Cell Science 1990, 97, 101. Tucker, J.B.; Milner, M.J.; Currie, D.A.; Muir, J.W.; Forrest, D.A.; Spencer, M.J. Eur. J. Cell Biol. 1986, 41,279. Dye, R.B.; Fink, S.P.; Williams, R.C.J. Biol. Chem. 1993, 268, 6847. Rowinsky, E.K.; Donehower, R.C.; Jones, R.J.; Tucker, R.W. Cancer Res. 1988, 48, 4093. Roberts, J.R.; Rowinsky, E.K.; Donehower, R.C.; Robertson, J.; Allison, D.C.J. Histochem. Cytochem. 1989, 37, 1659. Rowinsky, E.K.; Donehower, R.C. Pharmacol. Ther. 1991, 52, 35. Rowinsky, E.K.; Cazenave, L.A.; Donehower, R.C.J. Natl. Cancer Inst. 1990, 82, 1247. Roberts, J.R.; Allison, D.C.; Donehower, R.C.; Rowinsky, E.K. Cancer Res. 1990, 50, 710. Rowinsky, E.K.; Burke, P.J.; Karp, J.E.; Tucker, R.W.; Ettinger, D.S.;

334 Donehower, R.C. Cancer Res. 1989, 49, 4640. Turner, P.F.; Margolis, R.L.J. Cell Biol. 1984, 99,940. Lewis, S.A.; Ivanov, I.E.; Lee, G-H.; Cowan, N.J. Nature 1989, 342,498. Lewis, S.A.; Cowan, N.J. Nature 1990, 345, 674. Umeyama, T.; Okabe, S.; Kanai, Y.; Hirokawa, N. J. Cell Biol. 1993, 20, 451. 80. Gottesman, M.M.; Pastan, I. Ann. Rev. Biochem. 1993, 62, 385. 81. Borst, P.; Schinkel, A.H.; Smit, J.J.M.; Wagenaar, E.; VanDeemter, L.; Smith, A.J.; Eijdems, E.W.H.M.; Baas, F.; Zaman, G.J.R. Pharmacol. Ther. 1992, 60, 289. 82. Roy, S.N.; Horwitz, S.B. Cancer Res. 1985, 45, 3856. 83. Greenberger, L.M.; Lothstein, L.; Williams, S.S.; Horwitz, S.B. Proc. Natl. Acad. Sci. USA 1988, 85, 3762. 4. Greenberger, L.M.; Williams, S.S.; Horwitz, S . B . J . Biol. Chem. 1987, 262, 13685. 85. Jachez, B.; Nordmann, R.; Loor, F. J. Natl. Cancer Inst. 1993, 85, 478. 86. Chervinsky, D.S.; Brecher, M.L.; Hoelcle, M.J. Anticancer Res. 1993, 13, 93. 87. Balasubramanian, S.V.; Straubinger, R.M. Biochemistry 1994, 33, 8941. 88. Hockenbery, D. et al. Cell 1993,74, 957. 89. Miyashita, T.; Reed, J.C. Blood 1993, 81,151. 90. Bhalla, K., Huang, Y.; Tang, C.; Self, S. et al. Leukemia 1994, 8, 465. 91. Willingham, M.C.; Bhalla, K. J. Histochem. Cytochem. 1994, 42,441. 92. Cabral, F.; Barlow, S.B. FASEB J. 1989, 3, 1593. 93. Cabral, F.; Barlow, S.B. Pharmacol. Ther. 1991, 52, 159. 94. Sawada, T.; Cabral, F. J. Biol. Chem. 1989, 264, 3013. 95. Schibler, M.J.; Cabral, F. J. Cell Biol. 1986, 102, 1522. 96. Cabral, F. J. Cell Biol. 1983, 97, 22. 97. Cabral, F.; Wible, L.; Brenner, S.; Brinkley, B.R.J. Cell Biol. 1983, 97, 30. 98. Cabral, F.; Sobel, M.E.; Gottesman, M.M. Cell 1980, 20, 29. 99. McIntosh, J.R. In Microtubules; Hyams, J.S.; Lloyd, C.W., Eds. New York: Wiley-Liss, Inc., 1994; p.413. 100. Hayden, J.H.; Bowser, S.S.; Rieder, C.L.J. Cell Biol. 1990, 111, 1039. 101. Mitchison, T.J.; Salmon, E.D.J. Cell Biol. 1992, 119, 569. 102. Diaz, J.F.; Andreu, J.M. Biochemistry 1993, 32, 2747. 103. Ding, A.; Porteu, F.; Sanchez, E.; Nathan, C.F. Science 1990, 248, 370. 76. 77. 78. 79.

335 104. Manthey, C.L.; Brandes, M.E.; Perera, P-Y.; Vogel, S.N.J. Immunology 1992, 149, 2459. 105. Manthey, C.L.; Qureshi, N.; Stutz, P.L.; Vogel, S.N.J. Exper. Medicine 1993, 178, 695. 106. Lorsbach, R.B.; Murphy, W.J.; Lowenstein, C.J.; Snyder, S.H.; Russell, S.W.J. Biol. Chem. 1993, 268, 1908. 107. Manthey, C.L.; Perera, P-Y.; Salkowski, C.A.; Vogel, S.N.J. Immunology 1994, 152, 825. 108. Ding, A.; Sanchez, E.; Nathan, C.F.J. Immunology 1993, 151, 5596. 109. Farrar, M.A.; Schreiber, R.D. Ann. Rev. Immunol. 1992, 11,571. 110. Boulet, J.; Ralph, S.; Stanley, E.; Lock, P.; Dunn, A.R.; Green, S.P.; Phillips, W.A. Oncogene 1992, 7, 703.

337

SUBJECT INDEX Abeotaxanes Acylations, of taxoids Aldol reaction Amentotaxus

Anhydrotaxinol Artifacts, isolation Austrotaxus

Baccatins NMR X-ray I,II III IV,V VI VII 10-deacetyl, III 1-hydroxy, I 14-hydroxy- 10-deacetyl bcl-2

Biliary excretion, of taxoids Brevifoliol Biogenesis, of taxoids Cardiotoxicity, of taxoids Clemeolide

20,22,23,45,57-62,86 23,25,174,179,182-187,203,212 275-277 8 34,36 38 8 15 93 94 16 16,95,97,165-255 16 16,61,62,77 16 16 73 75 323-325 154 15,85 25

Colchicine Conformation, of side chain Cremophor EL 3,11-Cyclotaxanes Cytochrome 450

43 27 323 306-314 293 104-110 20,22 143-51

Deacylations, of taxanes Dehydration, of taxoids Deoxygenations, of taxoids Disposition, of taxoids

171-173,177-181 198-201,207 176,188,189,196,208-211 152

Enzymes, in side chain synthesis 7-Epimers, metabolism

277-281 133,195

Fluorination, of taxoids Formulation, of taxoids

197-201 103-30

Glucuroconjugates, of Taxol Glycosidation, of taxoids

142 25

c-myc

338 Hydrolysis, metabolic Hypersensitivity, to Taxol

136 109

g-Lactams Linkers, self-immolating Liposomes LPS

267-274 117-24 125 328

MAPs Mass Spectra, of taxoids Michael reaction Microtubules bundles treadmilling Multi-drug Resistance

303,318,321,329 55 266 301-332 311,317-319 304 313,322-325

NMR Spectra, of taxoids Numbering, of taxoids

58-91 13

Oxazolidines Oxazolines Oxetane, reactions Oxidation, metabolic of Taxol

260-262 280 171,175,189-193

P-glycoprotein Paleotaxus grandis Paleotaxus jurassica Paleotaxus rediviva Plasma binding, of taxoids Prodrugs, of Taxol Pro-prodrugs, of Taxol Pseudotaxus

325 7 7 7 152 110-24 117-24 8

Reduction, of taxoids Representations, of taxoids Ring contraction, A, of taxoids

204,205,210 13 42,170

134-7 202

Skeletal rearrangements, of taxoids 32,222-4 Solubility, of Taxol 106 Spicaledonine 22 Sulfation, metabolic 142 Sulfinimines 273-274 Taiwanxan Tau Taxacultins Taxagifine

15,93 303 17 15,22,27,65,93,96

339 Taxchins Taxchinins Taxicins Taxines biology NMR spectra structures Taxinines A B, E, H, J, K, L M Taxomyces andreanae Taxus Taxus baccata Taxus brevifolia Taxus celibica Taxus cuspidata Taxus globosa Taxus wallichiana Taxus yunnanensis Taxusin Taxuyunnanines

43,44 84,89 10,11,16 12,16,20,34,72,78,80-83 17,94 17 17,87 31 8,45 7,8,9,39 9,31,45,105 8,9 9 9 9 8 93 16,27

Torreya Tubulin isoforms isotypes

8,45 301-332 315 315

Urinary excretion, of Taxol UV Spectra, of taxoids

154 56-57

Verticilloids Vinblastine

57 306,314

Winterstein esters

11,24,31,41

16,27 16,31,94 79