Taxus brevifolia and Taxol Paclitaxel

A note on nomenclature: the name taxol was given to a diterpene ester with anticancer properties when it was first isolated in 1971 from Taxus brevifolia. When the compound was subsequently exploited commercially as a drug, Taxol was registered as a trademark. Accordingly, the generic name paclitaxel has been assigned to the compound. The literature now contains an unhappy mixture of the two names, though the original name taxol is most often employed.

" (Continued)

The anticancer drug taxol (Figure 5.43) is extracted from the bark of the Pacific yew, Taxus brevifolia (Taxaceae), a slow growing shrub/tree found in the forests of North-West Canada (British Columbia) and the USA (Washington, Oregon, Montana, Idaho, and North California). Although the plant is not rare, it does not form thick populations, and needs to be mature (about 100 years old) to be large enough for exploitation of its bark. The wood of T. brevifolia is not suitable for timber, and in some areas, plants have been systematically destroyed to allow cultivation of faster-growing commercially exploitable conifers. Harvesting is now strictly regulated, but it is realized that this will not provide a satisfactory long term supply of the drug. The bark from about three mature 100-year-old trees is required to provide one gram of taxol, and a course of treatment may need 2 grams of the drug. Current demand for taxol is in the region of 100-200 kg per annum.

All parts of Taxus brevifolia contain a wide range of diterpenoid derivatives termed taxanes, which are structurally related to the toxic constituents found in other Taxus species, e.g. the common yew, Taxus baccata. Over a hundred taxanes have been characterized from various Taxus species, and taxol is a member of a small group of compounds possessing a four-membered oxetane ring and a complex ester side-chain in their structures, both of which are essential for antitumour activity. Taxol is found predominantly in the bark of T. brevifolia, but in relatively low amounts (about 0.01 -0.02%). Up to 0.033% of taxol has been recorded in some samples of leaves and twigs, but generally the taxol content is much lower than in the bark. The content of some other taxane derivatives in the bark is considerably higher, e.g. up to 0.2% baccatin III (Figure 5.44). Other taxane derivatives characterized include 10-deacetyltaxol, 10-deacetylbaccatin III, cephalomannine and 10-deacetylcephalomannine. A more satisfactory solution currently exploited for the supply of taxol and derivatives for drug use is to produce these compounds by semi-synthesis from more accessible structurally related materials. Both baccatin III and 10-deacetylbaccatin III (Figure 5.44) have been efficiently transformed into taxol. 10-Deacetylbaccatin III is readily extracted from the leaves and twigs of Taxus baccata, and, although the content is variable, it is generally present at much higher levels (up to 0.2%) than taxol can be found in T. brevifolia. Taxus baccata, the common yew, is widely planted as an ornamental tree in Europe and the USA and is much faster growing than the Pacific yew. Cell cultures of T. baccata also offer excellent potential for production of taxol or 10-deacetylbaccatin III but are not yet economic; taxol yields of up to 0.2% dry weight

Taxol Production

R = Ac, baccatin III R = H, 10-deacetylbaccatin III

R = Ac, cephalomannine R = H, 10-deacetylcephalomannine docetaxel (taxotere)

R = Ac, baccatin III R = H, 10-deacetylbaccatin III

R = Ac, cephalomannine R = H, 10-deacetylcephalomannine docetaxel (taxotere)

cultured cells have been obtained. The use of microorganisms and enzymes to specifically hydrolyse ester groups from the mixture of structurally related taxanes in crude extracts and thus improve the yields of 10-deacetylbaccatin III has also been reported.

There is further optimism for new methods of obtaining taxol by microbial culture. Thus, a fungus, Taxomyces adreanae, isolated from the inner bark of Taxus brevifolia appears to have inherited the necessary genes from the tree (or vice versa) and is able to synthesize taxol and other taxanes in culture, though at only very low levels (20-50 ng l-1). A fungus, Pestalotiopsis microspora, recently isolated from the inner bark of the Himalayan yew (Taxus wallachiana) produces higher levels (60-70 |ig l-1), and if this could be optimized further it might form the basis for commercial production.

Paclitaxel (Taxol®) is being used clinically in the treatment of ovarian and breast cancers, non-small-cell lung cancer, small-cell lung cancer, and cancers of the head and neck. Docetaxel (Taxotere®) (Figure 5.44) is a side-chain analogue of taxol, which has also been produced by semi-synthesis from 10-deacetylbaccatin III. It has improved water-solubility compared with taxol, and is being used clinically against ovarian and breast cancers. Taxol acts as an antimitotic by binding to microtubules, promoting their assembly from tubulin, and stabilizing them against depolymerization during cell division. The resultant abnormal tubulin-microtubule equilibrium disrupts the normal mitotic spindle apparatus and blocks cell proliferation. Taxol thus has a different mechanism of action to other antimitotics such as vincristine (see page 356) or podophyllotoxin (see page 136), which bind to the protein tubulin in the mitotic spindle, preventing polymerization and assembly into microtubules. Taxol has also been shown to bind to a second target, a protein which normally blocks the process of apoptosis (cell death). Inhibition of this protein allows apoptosis to proceed.

The latex of some plants in the genus Euphorbia (Euphorbiaceae) is toxic, and can cause poisoning in humans and animals, skin dermatitis, cell proliferation, and tumour promotion (co-carcinogen activity). Many species of Euphorbia are regarded as potentially toxic, and the latex can produce severe irritant effects, especially on mucous membranes and the eye. Most of the biological effects are due to diterpene esters, e.g. esters of phor-bol (Figure 5.45), which activate protein kinase C, an important and widely distributed enzyme responsible for phosphorylating many biochemical entities. The permanent activation of protein kinase C is thought to lead to the uncontrolled cancerous growth. The most commonly encountered ester of phorbol is 12-0 -myristoylphorbol 13-acetate (Figure 5.45). The origins of phorbol are not fully delineated, but may be rationalized as in Figure 5.45. Cyclization of GGPP generates a cation containing a 14-membered ring system. Loss of a proton via cyclopropane ring formation leads to casbene, an antifungal metabolite produced by the castor oil plant, Ricinus communis (Euphorbiaceae). Casbene, via the ring closures shown in Figure 5.45, is then likely to be the precursor of the phorbol ring system.

In contrast to the cyclization mechanisms shown in Figures 5.43 and 5.45, where loss of diphos-phate generates the initial carbocation, many of the natural diterpenes have arisen by a different mechanism. Carbocation formation is initiated by protonation of the double bond at the head of the chain leading to a first cyclization sequence. Loss of the diphosphate later on also produces a carbocation and facilitates further cyclization. The early part of the sequence resembles that involved in hopanoid biosynthesis (see page 218), and to some extent triterpenoid and steroid biosynthesis (see page 214), though in the latter cases opening of the epoxide ring of the precursor squa-lene oxide is responsible for generation of the cationic intermediates. Protonation of GGPP can initiate a concerted cyclization sequence, terminated by loss of a proton from a methyl, yielding copalyl PP (Figure 5.46, a). The stereochemistry in this product is controlled by the folding of the substrate on the enzyme surface, though an alternative folding can lead to labdadienyl PP, electrophilic addition gives tertiary cation formation of cyclopropane ring with loss of proton

GGPP

GGPP

casbene

CH3(CH2)12 O OAc

12-O-myristoylphorbol 13-acetate esterifications

12-O-myristoylphorbol 13-acetate

* various O OH ) oxygenations HO phorbol casbene proposed | ring closures j esterifications

Figure 5.45

proposed | ring closures j

Figure 5.45

protonation of double bond gives tertiary carbocation; this allows concerted series of cyclizations terminating in proton loss

GGPP

GGPP

GGPP

Figure 5.46

GGPP

labdadienyl PP = (+)-copalyl PP

labdadienyl PP = (+)-copalyl PP

Figure 5.46

the enantiomeric product having opposite configurations at the newly generated chiral centres (Figure 5.46, b). From copalyl PP, a sequence of cyclizations and a rearrangement, all catalysed by a single enzyme, leads to ent-kaurene (Figure 5.47). As shown, this involves loss of the diphosphate leaving group enabling carbocation-mediated formation of the third ring system, and subsequent production of the fourth ring. Then follows a Wagner-Meerwein migration, effectively contracting the original six-membered ring to a five-membered one, whilst expanding the five-membered ring to give a six-membered ring. The driving force is transformation of a secondary carbocation to give a tertiary one, but this also results in the methyl group no longer being at a bridgehead, and what appears at first glance to be merely a confusing change in stereochemistry. Loss of a proton from this methyl generates the exocyclic double bond of ent-kaurene and provides an exit from the carbo-cationic system. The prefix ent is used to indicate enantiomeric; the most common stereochemistry

copalyl PP

loss of diphosphate generates allylic action; further cyclization of alkene on to cation copalyl PP

CO2H ent-kaurenoic acid sequential oxidation of methyl to carboxyl cyclization of alkene on to cation produces secondary cation sequential oxidation of methyl to carboxyl

ent-kaurene loss of proton generates alkene

O2 NADPH ,OH

CO2H ent-kaurenoic acid

O2 NADPH ,OH

3 x UDPGlc

H CO2H

steviol

ent-kaurene

^OGlc —Glc loss of proton generates alkene

Steviol Rearrangement

secondary cation converted into tertiary cation by 1,2-alkyl migration

secondary cation converted into tertiary cation by 1,2-alkyl migration

3 x UDPGlc

H CO2H

steviol

CO2Glc stevioside

CO2Glc stevioside

CO2H

steviol

CO2H

steviol

Figure 5.47

is that found in labdadienyl PP (Figure 5.46) and derivatives, so the kaurene series is termed enan-tiomeric.

ent-Kaurene is the precursor of stevioside (Figure 5.47) in the plant Stevia rebaudiana (Com-positae/Asteraceae) by relatively simple hydroxy-lation, oxidation, and glucosylation reactions. Both glucosyl ester and glucoside linkages are present in stevioside, which help to confer an intensely sweet taste to this and related compounds. Stevio-side is present in the plant leaf in quite large amounts (3 -10%), is some 100 - 300 times as sweet as sucrose, and is being used commercially as a sweetening agent.

The alternative stereochemistry typified by lab-dadienyl PP can be seen in the structure of abi-etic acid (Figure 5.48), the major component of the rosin fraction of turpentine from pines and other conifers (Table 5.1). Initially, the tricyclic system is built up as in the pathway to ent-kaurene (Figure 5.47), via the same mechanism, but generating the enantiomeric series of compounds. The cation loses a proton to give sandaracopimara-diene (Figure 5.48), which undergoes a methyl migration to modify the side-chain, and further proton loss to form the diene abietadiene. Abietic acid results from sequential oxidation of the 4a-methyl. Wounding of pine trees leads to an accumulation at the wound site of both monoterpenes and diterpenes, which may be fractionated by distillation to give turpentine oil and rosin (Table 5.1). The volatile monoterpenes seem to act as a solvent to allow deposition of the rosin layer to seal the wound. The diterpenes in rosin have both anti-fungal and insecticidal properties.

Extensive modification of the labdadienyl diter-pene skeleton is responsible for generation of the ginkgolides, highly oxidized diterpene trilac-tones which are the active principles of Ginkgo biloba * (Ginkgoaceae). Several rearrangements, ring cleavage, and formation of lactone rings can broadly explain its origin (Figure 5.49), though this scheme is highly speculative and likely to be incorrect. Although detailed evidence is lacking, it is known that labdadienyl PP is a precursor, and most probably dehydroabietane also. The unusual tert-butyl substituent arises as a consequence of the A ring cleavage. Bilobalide labdadienyl PP

labdadienyl PP

loss of proton generates alkene

loss of proton generates alkene

protonation of alkene allows W-M 1,2-methyl shift

protonation of alkene allows W-M 1,2-methyl shift sandaracopimarenyl cation

(-)-sandaracopimaradiene sandaracopimarenyl cation

(-)-sandaracopimaradiene

Figure 5.48

abietenyl cation

Figure 5.48

labdadienyl PP

Figure 5.48

Figure 5.48

abietenyl cation labdadienyl PP

A ring cleavage further sequence of oxidative reactions

abietenyl cation

A ring cleavage

W-M V 1,2-alkyl shift

ginkgolides dehydroabietane a a ho2c ring closures M (1 hemiacetal, 3 lactones)

OH y as indicated CÛ2H O

W-M V 1,2-alkyl shift ginkgolides are usually drawn as if viewed from the other side

1,2-alkyl shift loss of carbons as indicated; lactone formation from residual carboxyl and alcohol functions O

co2h OH 12

"O

ginkgolide

Figure 5.49

"O

ginkgolide

e.g. bilobalide

Figure 5.49

(Figure 5.49) contains a related C15-skeleton, and is most likely a partially degraded ginkgolide. Ginkgo is the world's oldest tree species, and its leaves are now a currently fashionable health supplement, taken in the hope that it can delay some of the degeneration of the faculties normally experienced in old age.

In forskolin (Figure 5.51), the third ring is heterocyclic rather than carbocyclic. The basic skeleton of forskolin can be viewed as the result

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  • Larry
    Where is taxol present in the commomn yew?
    8 years ago

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