Minggu, 11 Oktober 2009

Taxol: A Case Study in Natural Products Chemistry


In the late 1950s the National Cancer Institute announced a new program aimed at screening plant extracts for chemotherapeutic activity. As a direct result of this program, extracts from the bark of the pacific yew, Taxus brevifolia, were shown to inhibit tumor growth. In 1969 the most active component of the extract was isolated. Its structure was published in 1971. The compound was named taxol. In 1983 the National Cancer Institute began clinical trials of taxol's safety and effectiveness against various types of cancer. In 1992 the Food and Drug Administration approved the use of taxol for treatment-resistant ovarian cancers, and in 1994 the FDA also approved taxol for recurrent breast cancer chemotherapy. Figure 1 presents the structure of taxol.

Figure 1

Taxol: A Wonder Drug?

Taxol's Mode of Action

Many natural products display cytotoxic activity. What piqued interest in taxol was that it killed cells by a different mechanism than other drugs. During cell mitosis, a cellular protein called tubulin undergoes a reversible polymerization to form structures known as microtubules, which are required for the formation of the mitotic spindle. Taxol binds to the microtubules, stabilizing them, and altering the tubulin-microtubule equilibrium. As a result, the cells cannot undergo normal mitosis and they die.

Cost-Benefit Analysis

The concentration of taxol in the the Pacific yew is approximately 100 mg per kilogram of bark (0.01%). The clinical trials that the NCI performed in 1989 required 27,000 kg of bark. Since a typical 100 year old yew yields about 3 kg of bark, these trials required 9,000 trees. Since collection of the bark kills the tree, the use of taxol from natural sources threatened the species with extinction. Fortunately, it was discovered that a structurally related compound, 10-deacetylbaccatin III, can be extracted from the needles and leaves of the European yew, Taxus baccata, in approximately 0.1% yield. This does not require killing the tree. The structure of 10-deacetylbaccatin III is shown in Figure 2.

Figure 2

A Saviour in Our Midst

Syntheses of Taxol

The availability of 10-deacetylbaccatin III made it an attractive candidate for preparing taxol. One successful synthesis is outlined in Figure 3.

Figure 3

A "Semi-Synthesis" of Taxol

This "semi-synthetic" approach produced taxol in over 50% yield from 10-deacetylbaccatin III. At least two pharmaceutical companies now uses similar methodology for the commercial production of taxol from 10-deacetylbaccatin III. A number of companies have developed processes for producing taxol from cell cultures of Taxus brevifolia, Taxus cuspidata, and Taxus canadensis.

While the preparation of taxol from a precursor such as 10-deacetylbaccatin III has obvious commercial advantages, synthetic organic chemists focus on the synthesis of such targets from simpler molecules. They see compounds like taxol as mountains to be climbed simply because they are there. In 1994, two research groups reported total syntheses of taxol. We will look at several aspects of each synthesis as illustrations of basic chemical principles that we have discussed or will be discussing in the future.

The first report of a total synthesis of taxol appeared in the Journal of the American Chemical Society 1994, 116, 1597-1598. The authors of the article were Robert A. Holton, Carmen Somoza, Hyeong-Baik Kim, Feng Liang, Ronald J. Biediger, P. Douglas Boatman, Mitsuru Shindo, Chase C. Smith, Soekchan Kim, Hossain Nadizadeh;, Yukio Suzuki, Chunlin Tao, Phong Vu, Suhan Tang, Pingsheng Zhang, Krishna K. Murthi, Lisa N. Gentile, and Jyanwei H. Liu. In addition to many authors, there are many interesting aspects to this paper. We will focus on three:

  • the use of a chiral starting material
  • the development of synthetic methodology
  • the application of conformational analysis

Figure 4 shows the start and finish of the synthesis of taxol and identifies the atoms in the starting material with those in the product by color coding.

Figure 4

From Start to Finish

Using a chiral starting material insured that the taxol would be optically active rather than a racemic mixture. The presence of the chiral centers in (-)-camphor insured the production of an enantiomeric excess whenever a new chiral center was generated during the synthesis.

A key intermediate in this synthesis was the bicyclic compound 6 which was produced by a procedure known as the "epoxy alcohol fragmentation" as diagrammed in Figure 5.

Figure 5

Coming Unglued

This fragmentation reaction had been developed 10 years earlier as part of earlier investigations into the synthesis of the "taxane skeleton", i.e.the carbon framework of taxol and compounds structurally related to taxol. The elaboration of structure 6 to taxol required knowledge of the shape of the compound, i.e. its conformation.

According to 1H-NMR studies, this bicyclic ring system exists in four different conformations as shown in Figure 6 with the chair-boat conformation most stable and the chair-chair and boat-chair conformations about 2.5 kcal/mol higher in energy.

Figure 6

Boats and Chairs

Knowing what the conformation of the material was allowed the investigators to develop a rational design to subsequent steps in the synthesis.

The overal yield of taxol starting from (-)-camphor was 0.1%!!!

Structure-Activity Correlations

While the mechanism of taxol's cytotoxicity is fairly well understood, it is not clear what the minimum structural features of the molecule are that will still prevent cell division. One approach to this question involves determination of the cytotoxicity of compounds in which various functional groups in taxol have been modified. Figure 7 shows the structures of four compounds where seemingly minor changes in the functionality hav

Figure 7

Investigating Cause and Effect

Replacement of the benzoate group at C-2 with a hydrogen atom produced a derivative with greatly reduced in vitro cytotoxic activity. In contrast, the cytotoxicity of the compound produced by replacement of the OH group at C-7 with a hydrogen atom was 40 times greater than that of taxol. Reduction of the C-9 carbonyl group yielded an alcohol that was slightly more active than taxol, replacement of the ester group at C-10 with hydrogen formed a derivative that was slightly less active. Investigations of this type are necessary to understand the mode of action of taxol at the molecular level.

Once you've made a taxol derivative, how do you test its activity? The standard methods involve in vivo and in vitro tests. In an in vitro test, the effect of the derivative on the growth of cell cultures is observed. In in vivo tests the compound is administered to test animals with specific types of malignancies. The survival rate of the injected animals is compared to that of controls.

An interesting spectroscopic method was recently described in an article entitled "Taxol and Taxotere: Discovery, Chemistry, and Structure-Activity Relationships" by Daniel Guenard, Francoise Gueritte-Voegelein, and Pierre Potier in Accounts of Chemical Research 1993, 26, 160-167. The method is based on the fact that the UV absorbance of a solution that contains a tubulin-microtubule mixture changes as the composition of the mixture changes. The composition of the mixture, in turn, changes with temperature. At 37o tubulins assemble into microtubules, while at 0o the microtubules disassemble back to the tubulins. Figurre 8 shows a typical UV spectrum.

Figure 8

An Activity Assay

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