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Chapter 10

Taxol, an Exciting Anticancer Drug from Taxus brevifolia A n Overview

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David G. I. Kingston Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0212 The taxane diterpenoid taxol was first reported in 1971, but it has only recently been recognized as a highly effective anticancer drug. The history of taxol's development is reviewed with an emphasis on the problems that almost prevented the discovery of its clinical activity, and on the key factors that kept it under investigation. Recent research on the structure-activity relationships and the synthesis of taxol is also reviewed The toxic properties of yew have been known for at least two thousand years. Thus, Julius Caesar recorded that Catuvolcus, king of the Eburenes, poisoned himself with yew rather than face capture by Caesar's legions (7), and many other early accounts of yew poisoning have been reviewed (2). Because of this, initial chemical studies of the constituents of yew (either the English yew, Taxus baccata, or the Japanese yew, Taxus cuspidata) concentrated primarily on its toxic principles, culminating in the structure elucidation of the first taxane diterpenoids in the 1960's. It is thus ironic that the yew, long known as a tree of death, should become the source of one of the most promising and important new anticancer drugs of the last twenty years. Discovery, Isolation, and Structure Elucidation of Taxol By 1960, the National Cancer Institute (NCI) had recognized the importance of natural products as potential anticancer drugs, largely through the leadership of the late Jonathan Hartwell and his work on the constituents of Podophyllum peltatum. Under his guidance, a contract program was established to collect plant materials, screen them for biological activity, andfractionatethem to obtain the pure active compounds. The plant collection was conducted by a team at the U.S. Department of Agriculture (USDA) under Dr. Robert Perdue, Jr., and one of the fractionation contracts was awarded to a team at the newly established Research Triangle Institute in North Carolina under Dr. Monroe Wall. One day in 1964, Dr. Wall received a sample of the bark of the western yew, Taxus brevifolia,fromNCI, collected by the USDA botanists. This sample was one of hundreds received that year, but this one was unusual in that its extracts showed a high cytotoxicity in the KB cell culture assay, although this activity was not exceptional. However, T. brevifolia bark was selected forfractionation,and a large recollection of bark was received in 1966. Fractionation of the bark extract proved

0097-6156/93/0534-O138$06.00/0 © 1993 American Chemical Society

In Human Medicinal Agents from Plants; Kinghorn, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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difficult because it turned out that the acdve material was present in very low abundance, and it was not until 1969 that adequate quantities of the active compound became available for structural work. The structure elucidation of the active compound, named taxol because of its botanical origin and the fact that it had a hydroxyl group, proved to be a challenging task. Initial studies were complicated by the difficulty of obtaining reliable mass spectrometry data, and by the failure of taxol to crystallize in a form suitable for Xray analysis. The structure was finally solved when Dr. Mansukh Wani at RTI discovered that it was possible to cleave taxol into two portions by Zemplen methanolysis. He was then able to obtain crystalline derivatives of each fragment. The structures of these two derivatives were elucidated by means of X-ray crystallography by Dr. Andrew McPhail at Duke University as the p-bromobenzoate derivative, 1, and the bisiodoacetate 2. The structure of taxol was then determined by oxidation studies with maganese dioxide, which established that the side chain

PhCOO

1

2

esterifies the taxane diterpenoid unit at the allylic hydroxyl group. The structure of taxol, first published in 1971, is thus 3 (5). It belongs to the class of taxane

3 diterpenoids, or taxoids, and is structurally related to the toxic constituents of yew such as taxine B (4).

4

In Human Medicinal Agents from Plants; Kinghorn, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Development of Taxol as an Antitumor Agent At the time of its discovery, taxol was but one of a number of promising natural product leads that were being investigated. The Wall group had earlier isolated camptothecin (4), and his and other research groups were reporting new active compounds on a regular basis. Taxol showed clear but modest in vivo activity in the P-388 and L-1210 leukemia assays, and its activity in these assays was no better than that of various other compounds competing for limited development resources, and activity against leukemias was not in and of itself sufficient cause for development In addition, taxol had two significant disadvantages. The first disadvantage was that of supply. Taxol was obtained from T. brevifolia bark by a laborious isolation procedure in an overall yield of only 0.02% w/w (5). Although T. brevifolia is not a rare tree, and was until recently treated as trash and burned by timber harvesters in the Pacific Northwest, it was nevertheless clear from the very first studies on taxol that its use as an anticancer drug would present a massive supply problem To put this into perspective, the bark of about three trees is required to produce one gram of taxol. An early unpublished survey carried out by the National Cancer Institute (Dr. M . Suffness, personal communication) showed that the bark of T. brevifolia was the best source of biological activity, and thus there was little hope that a more abundant plant part might yield adequate amounts of taxol. The second major disadvantage was solubility. Because of their narrow therapeutic index, anticancer drugs are almost all administered by intravenous infusion, and taxol is inactive orally against mouse tumors. A water-soluble formulation is thus necessary. Taxol is, however, almost completely insoluble in water, and it was not immediately obvious that a satisfactory water-soluble formulation could be achieved. In due course, a solution to this problem was eventually found, as described below, but at the cost of very nearly ending taxol's development. Because of these two major disadvantages, the development of taxol as an anticancer drug was not pursued aggressively during the early 1970's. However, various new in vivo assay systems were developed during this time, including the use of athymic mice as hosts for human tumor xenografts. The NCI was thus able to test promising compounds against several new assays such as mammary, lung, and colon xenografts, and the B16 mouse melanoma assay. Among the compounds tested in this way was taxol, and the results were highly encouraging; taxol showed clear and convincing activity against various human solid tumors, including the MX-1 mammary xenograft, and against the B16 mouse melanoma (Table 1) (5). Based on these assay results, the decision was made in 1977 to begin the development of taxol as an anticancer agent Interest in taxol received a significant boost from the discovery by Susan Horwitz in 1979 that it promoted the assembly of tubulin into stable microtubules (6). Tubulin is a ubiquitous cellular protein that is intimately involved in the mitotic process. During mitosis, tubulin, which exists in a- and 0-fonns, reversibly assembles to form hollow microtubules, and the chromosomes separate with the assistance of these microtubules. After mitosis, the microtubules disassemble to regenerate tubulin. Several drugs, including the important anticancer drugs vinblastine and vincristine, are believed to exert their effects by preventing the assembly of tubulin into microtubules (7). Taxol, however, promotes the assembly of tubulin into heat- and calcium-stable microtubules, and this presumably prevents cellular division and facilitates cell death. Taxol binds stoichiometrically and noncovalently to tubulin, but the binding site is on the assembled microtubule rather than the tubulin sub-unit (8).

In Human Medicinal Agents from Plants; Kinghorn, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Taxol, an Exciting Anticancer Drug

Table 1. Selected Antitumor Activity Data for Taxol

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Tumor system

Route Regimen Optimal dose T/C(%) Evaluation (mglkglinj) atOD tumor/drug ++ B16 melanoma 283 5 Dailyx9 ++ P1534 leukemia 3.75 300 Daily x 10 ipAp + P388 leukemia 170 43 q4dx3 + L1210 leukemia ipAp 131 20 Daily x 15 + Colon 26 ip/ip q4dx2 30 161 ++ MX-1 mammary (-67) src/sc 200 Daily x 10 xenograft + LX-1 lung src/sc 200 Daily x 10 (13) xenograft + CX-1 colon src/sc 400 Daily x 10 (12) xenograft During the period 1978-1982, various preclinical studies were carried out on taxol; the taxol required for these studies was obtained by large-scale isolation from T. brevifolia bark. As noted above, formulation of taxol was a difficult problem, and eventually a formulation was developed using Cremophor EL, a polyethoxylated castor oil, and absolute ethanol, the whole being diluted with 5% dextrose in water or normal saline before use (9). Toxicology studies were also performed, and LD50 values ranged from 34 mg/kg for rats to 9 mg/kg for beagle dogs (5). Phase I clinical trials were initiated in 1983, and these very nearly proved disastrous. Taxol must be given at relatively high doses [a typical course of treatment uses a dose of 250 mg/m (70)], and thus relatively large levels of Cremophor adjuvant must be administered. Some severe allergic reactions were observed during the Phase I clinical trials, including at least one death (77). These problems were probably due to the Cremophor adjuvant, since allergic reactions have been observed with this compound in other cases (72), but they very nearly halted further studies with taxol. Fortunately, the novelty of taxol's mechanism of action encouraged the clinicians to persevere, and the allergic reactions were minimized by premedication with glucocorticoids and antihistamines, and by lengthening the period of infusion. Phase I studies were then successfully completed, and in one study partial responses were observed in four of 12 patients with melanoma (75). The dose-limiting toxicity of taxol was found to be leukopenia, with other toxicities being neurotoxicity, nausea and vomiting, various allergic reactions, caidiotoxicity, and stomatitis. Phase II clinical trials were initiated in 1985. Although the extent of these trials has been limited by the availability of taxol, the results to date have been excellent, especially in comparison with other anticancer drugs. At the time of this writing, complete reports have appeared of results in ovarian and breast cancers. In ovarian cancer, a response rate of 30% was observed in a group of 40 patients, with one complete response (74). Particularly encouraging was the fact that these responses were noted in heavily pie-treated patients, many of whom were resistant to cisplatin. In breast cancer, with a group of 25 patients who had only one prior chemotherapy regimen, a response rate of 56% was observed, with 12% complete and 44% partial responses (70). These results, together with other as yet unpublished studies, clearly demonstrate the clinical effectiveness of taxol as an anticancer drug, and presage a bright future for it once the twin problems of supply and solubility have been overcome. 2

In Human Medicinal Agents from Plants; Kinghorn, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Enhancement of the Taxol Supply Because of the importance of increasing the taxol supply, various approaches have been adopted in addressing this problem Each will be discussed briefly.

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Isolation from T. brevifolia Bark. This approach is the most expeditious one for the production of taxol for clinical use, since it has already been approved by the Food and Drug Administration. Currently (1992) all taxol used for clinical purposes is obtained by this route, but the limited supply of T. brevifolia bark and the destruction of the tree that necessarily accompanies collection of it prevent this from being a viable long-term source of taxol. Isolation from Other Parts of Various Taxus Species. Several surveys of the taxoid content of various Taxus species have appeared; since the taxoids cephalomannine (5) and baccatin EQ (6) can be converted to taxol by procedures described below, their occurrence is of almost as much interest as is that of taxol. The major finding to emerge from this work is that the taxol content of Taxus leaves

5

6 7

R = Ac R=H

(or needles) is at least as great as that reportedfrombark. Thus Wheeler et al., found taxol contents up to 0.033% w/w in T. brevifolia shoots (needles plus twigs), and a maximum of 0.010% w/w in the bark (17). Significant variations in taxol content are seen depending on factors such as the season when collected, handling procedures, geographical location, and population. The content of other taxoids can be even greater; thus baccatin HI was observed at levels up to 0.2% w/w in T. brevifolia (77), and 10-deacetylbaccatin HI (7) can be obtained in yields of 0.1% w/wfromfresh leaves of T. baccata (18). Partial Synthesis from Baccatin III. Since baccatin III and 10-deacetylbaccatin i n both occur in good yield in the needles of Taxus spp., methods to convert them to taxol would be of significant importance in the overall approach to improving the taxol supply. Although the desired conversion is simply that of acylation at the C-13 hydroxyl group of baccatin HI with an appropriately substituted acid, this conversion is in fact difficult to carry out because of the very hindered nature of the C-13 hydroxyl group. Several approaches have, however, been devised to overcome this problem. The first approach, developed by Potier and his collaborators, involved acylation of a suitably protected baccatin i n with cinnamic acid. The cinnamoyl group was then functionalized by the Sharpless hydroxyamination procedure to yield taxol and various stereo- and regio-isomers (79). This approach is of limited utility because of the formation of these isomers, but it did have the importantfringebenefit of leading to the synthesis of the taxol analogue taxotere (8) from 10-deacetylbaccatin HI. This analogue shows a better activity than taxol in some assays (20), and is currently in clinical trials in France.

In Human Medicinal Agents from Plants; Kinghorn, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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The second approach, developed by Potter and Greene, involve a direct acylation of a protected baccatin m or 10-deacetylbaccatin m with a complete protected taxol side chain (18). The key reaction in this process proceeds in 80% yield at 50% conversion, and taxol can be obtained in 38% overall yield from 10-deacetylbaccatin m, assuming no recycling. The third general approach has been developed by Holton, who has shown that a suitably derivatized [J-lactam (9) will couple to a protected baccatin m (10) in excellent yield, as shown below (Holton, R.A., personal communication, 1990). OAc

Q

oSIEtj

Eto