WO2001034589A1

WO2001034589A1 - Semi-synthesis of baccatin iii from 9-dihydro-13-acetylbaccatin iii

Info

Publication number: WO2001034589A1
Application number: PCT/CA2000/001293
Authority: WO
Inventors: Lolita Zamir
Original assignee: Lolita Zamir
Priority date: 1999-11-05
Filing date: 2000-11-06
Publication date: 2001-05-17
Also published as: AU1260501A

Abstract

The present invention relates to a semi-synthetic process to convert a naturally occurring taxane into a suitable starting material for the synthesis of paclitaxel and related compounds. Specifically, the present invention relates to a process for the conversion of 9-dihydro-13-acetylbaccatin III into baccatin III which can then be used as starting material for the synthesis of taxane derivatives such as paclitaxel, docetaxel, cephalomannine and other taxanes structurally related to baccatin III.

Description

SEMI-SYNTHESIS OF BACCATIN III FROM 9-DIHYDRO-13-ACETYLBACCATIN III

FIELD OF THE INVENTION The present invention relates to a semi-synthetic process to convert a naturally occurring taxane into a suitable starting material for the synthesis of paclitaxel and related compounds. Specifically, the present invention relates to a process for the conversion of 9-dihydro-13- acetylbaccatin III into baccatin III which can then be used as starting material for the synthesis of taxane derivatives such as paclitaxel, docetaxel, cephalomannine and other taxanes structurally related to baccatin III.

BACKGROUND OF THE INVENTION

The taxane family of terpenes is considered to be an exceptionally promising group of cancer chemotherapeutic agents. Many taxane derivatives, including paclitaxel, docetaxel, taxcultine canadensol are highly cytotoxic and possess strong in vivo activities in a number of leukemic and other tumor systems. Paclitaxel, and a number of its derivatives, have been shown to be effective against advanced breast and ovarian cancers in clinical trials (W.P. MacGuire et al., Annals of Internal Medicine, vol 111, pg. 273, 1989). They have also exhibited promising activity against a number of other tumor types in preliminary investigations. Paclitaxel has recently been approved in the U.S. and Canada for the treatment of ovarian cancers (Rose et al, in "The Alkaloids", A. Brossi, Ed., Academic Press, New York, Paclitaxel: A Review of its preclinical in vivo Antitumor Activity. Anti-Cancer Drugs 3, 311-321 1992; and Sufifness, M., Paclitaxel: from discovery to therapeutic use. Ann. Rep. In Med. Chem., 28, 305-314, 1993). Taxanes are believed to exert their antiproliferative effect by inducing tubulin polymerization, which forms extremely stable and nonfunctional microtubules (Schiff, et al, Promotion of Microtubule Assembly in vitro by Paclitaxel. Nature, 277, 665-667, 1979). However, a major problem with the clinical studies is the limited availability of paclitaxel and its derivatives.

Taxanes are natural products which can be isolated from yew trees. The first taxane to be characterized was paclitaxel (also known as taxol™) which was isolated and purified from the bark of the Pacific yew in 1971. The only available natural source of paclitaxel to date are several species of a slow growing yew (genus Taxus), wherein paclitaxel is found in very low concentrations (less than 400 parts per million) in these trees. Furthermore the extraction is difficult, the process is expensive and the yield of paclitaxel is low (Huang et al, J. Nat. Prod. 49 665, 1986, reported a yield of 0.00025% of a crude paclitaxel fraction from Taxus brevifolia bark).

Paclitaxel

Paclitaxel can be isolated from the bark of Taxus brevifolia, the pacific yew tree, or from Taxus baccata, its European relative. Since removal of the bark destroys the trees and endangers the species, isolation of taxanes from the stems and needles of various Taxus species offers hope that the supply of taxanes, in particular paclitaxel, would become more abundant.

The preparation of paclitaxel derivatives, some of which have been reported to demonstrate enhanced chemotherapeutic activity, ultimately depends upon the supply of the parent compound - baccatin III. The structure of baccatin III has the basic diterpenoid structure of paclitaxel without the side chain at the C-13 position.

Baccatin III

Baccatin III is an important starting material in paclitaxel semi-synthesis Therefore the significance of baccatin III will likely increase as more clinical studies are performed using paclitaxel and its derivatives It appears that water soluble paclitaxel-like compounds, with slightly modified C-13 side chains, may be more desirable as cancer chemotherapeutic agents than the naturally occurring paclitaxel, which has lower water solubility This further increases the urgent need for a source of baccatin III as a starting material to synthesize both paclitaxel and second or third generation paclitaxel-like compounds In particular, there is a need for an improved method of isolating and/or synthesizing Baccatin III

The majority of research to date has been concerned with the development of techniques to increase the availability of either paclitaxel or baccatin III These techniques have included improvements to the isolation and purification processes (U S Patent 5,407,674 and U S Patent 5,380,916), to the total synthesis (U S Patent No 5,405,972) and partial synthesis (from more abundant paclitaxel precursors) and also isolation from a variety of cell culture systems (U S Patent No 5,019,504) In addition, an endophytic fungi isolated from bald cypress (Taxodium distichum) was reported to produce very small amounts of paclitaxel (Strobel, R et al , Microbiology, 142, 2223-2226, 1996)

Due to the structural complexity of paclitaxel, partial synthesis is a far more viable approach to providing adequate supplies of paclitaxel and paclitaxel precursors and derivatives than total synthesis The first successful semi-synthesis of paclitaxel was developed by Denis et al, (U S Patent No 4,924,011 re-issued as 34,277), using the starting material 10- deacetylbaccatin III which can be extracted in relatively high yield from the needles of specific species.

10-Deacetylbaccatin III

Most of the research to date, regarding the semi-synthesis of paclitaxel has involved 10- deacetylbaccatin III The conversion of 10-deacetylbaccatin III into paclitaxel is typically achieved by protecting the hydroxy at C-7, attachment of an acetyl group at the C-10 position, attachment of a C-13 β-amido ester side chain at the C-13 position through esterifi cation of the C-13 alcohol with the β-lactam moiety, and deprotecting C-7 Since the supply of 10-deacetylbaccatin III is limited, alternative semi-synthetic starting materials should be considered

Research has recently centred on semi-synthesis of paclitaxel from 10-deacetylbaccatin III because it is the major metabolite obtained from specific species of the European Yew (Taxus baccata) However to date, the yields of 10-deacetylbaccatin III have been unsatisfactory, ranging from 50-165 mg taxane per kilogram of starting material (i.e. providing yields of between 0 005 to 0 017%) Hence there is an urgent need for novel semi-synthetic techniques to produce higher yields of paclitaxel precursors, such as baccatin III, for subsequent use in the production of paclitaxel and paclitaxel derivatives Another taxane, 9- dihydro-13-acetylbaccatin, is produced as a major metabolite in a certain Taxus species and has been used as a paclitaxel precursor (Canadian Patent Application Nos 2,188,190 and 2,204,197) Relatively large amounts of a 7-protected baccatin III or baccatin III itself can be produced using the reported processes The significance of this recent work is that the

yield of 9-dihydro-13-acetylbaccatin III, which varies from 0 5 to 2.5 g per kilogram of dry plant depending on the collection site, is much higher than the yield of 10-deacetylbaccatin III that can be isolated from natural sources Using the 9-dihydrol3-acetylbaccatin III in the synthetic process of the Canadian Patent Application No 2,188,190 it was possible to obtain 7-protected baccatin III in 20% yield The synthetic steps required for this conversion included protecting the hydroxy group at the C-7 position or C-9 , or both C-7 and C-9 sequentially, oxidizing the resulting group at the C-9 position, and deacylating the ester at positions C-13 Baccatin III could then be obtained by deacylating the ester at the C-7 position

The present invention provides a new method of converting 9-dihydro-13-acetylbaccatin III into baccatin III which can then be used as a precursor to paclitaxel and paclitaxel intermediates and derivatives The method allows the production of relatively large amounts of baccatin III without the use of protecting groups at any step in the process

SUMMARY OF THE INVENTION

The present invention is directed towards a method of producing baccatin III, from 9- dihydro-13-acetylbaccatin III, which is a naturally occurring taxane produced in high yields in Taxus canadensis The baccatin III generated by this method, can then be used as a starting material for the synthesis of paclitaxel and paclitaxel intermediates and derivatives

Accordingly, it is an object of this invention to provide a reproducible method for the semi- synthesis of baccatin III from the naturally occurring compound, 9-dihydro-13-acetylbaccatin III, isolated from plant matter derived from the Taxus genus of plants

In accordance with an aspect of the present invention there is provided a method for the semi- synthesis of baccatin III, and other intermediates, that proceeds with higher yields than currently known methods In accordance with another aspect of the present invention there is provided a process for the preparation of Baccatin III from a compound of formula (X)

X

which comprises the steps of

(i) oxidizing the hydroxy groups, on a compound of Formula X, at the C-7 and C- 9 positions, (ii) reducing the resulting ketone at the C-7 position,

(iii) epimerizing the resulting 7α-hydroxy, and (iv) deacylating the ester at the C-13 position

In accordance with another aspect of the present invention there is provided compounds of the general Formula I

Formula I

wherein R is an alpha-hydroxy or ketone group.

In accordance with another aspect of the present invention there is provided compounds of the general Formula II.

Formula II

wherein Rl is a hydroxy or ketone group and R2 is a hydroxy or O-acetyl group. DETAILED DESCRIPTION OF INVENTION

The present invention relates to a high yield process for converting 9-dihydro- 13- acetylbaccatin III (an abundant taxane found in T. canadensis needles), into baccatin III, which can subsequently be used as starting material for the synthesis of paclitaxel and related compounds.

The starting material for use in this invention is vegetal material, selected from a group of plants commonly referred to as taxads. The most suitable plants of this group are the species Taxus. Amongst the Taxus species, Taxus canadensis is a preferred source for use in the semi-synthetic method claimed in the present invention and differs from other yews both in its physical appearance (it is a small ramping evergreen bush), and in the composition of some of its taxanes. Paclitaxel, cephalomannine and 10-deacetylbaccatin III can be isolated from Taxus canadensis and are also found in most, if not all, other yews. Taxus canadensis is, however, the only yew presently known to accumulate a significant quantity of 9-dihydro- 13-acetylbaccatin III in its needles, wherein it is found in concentrations 3 - 7 times greater than paclitaxel (Zamir L. O et al. Tetrahedron Letters 33, 5173, 1992). The only other yew where 9-dihydro-13- acetylbaccatin III is found albeit in traces is the Chinese yew (Taxus chinenesis) (Zhang, S.; Chen, W. M; Chen, Y. H. Yaoxue Xuebao 1992, 27, 268).

9-dihydro-13-acetylbaccatin III The methods disclosed herein are equally effective when using the roots or bark of the Taxus bushes but the preferred source is the needles, which are in abundant supply and one of the most renewable parts of the plant.

A number of different methods have described the isolation and purification of 9-dihydro- 13- acetylbaccatin III ( Zamire et al. Tetrahedron Lett. 1992, 33, 5173, Gunawardana G. P. et al, J. Nat. Prod. 1992, 55, 1686 and Zamir et al. Can. J. Chem. 1995, 73, 655,). 9-Dihydro-13- acetylbaccatin III is particularly useful as a synthetic starting material since it can be isolated from plant material using simple recrystallizations (Canadian Patent No. 2,213,952) instead of the numerous silica gel column and HPLC techniques commonly used for isolation of other starting materials, eg. 10-deacetylbaccatin III. In this way, 9-dihydro- 13 -acetylbaccatin III can be obtained in relatively high yield with relative ease, thereby rendering it an ideal starting material for many semi-synthetic pathways.

9-Dihydro- 13 -acetylbaccatin III, 1, is the major component isolated from the needles of the Canadian yew taxus canadensis, as referenced in J. Nat. Prod., 53: 1249, 1990; Tetrahedron Lett., 33: 5235, 1992; Tetrahedron Lett., 33: 5173, 1992; Can. J. Chem., 73: 655, 1995; Phytochemistry, 41: 803, 1996; J. Nat. Prod, 55: 1686, 1992. Typically 1.0 kg of dry needles will afford from 0.5 to 1.0 g of pure, isolated 9-dihydro- 13-acetylbaccatin III but can possibly vary up to ranges of 2.0 to 2.5 g per kg of dry plant material. Its efficient transformation to baccatin III, 2, would be beneficial since this would be another large source of starting material for the semi-synthesis of paclitaxel.

13

The major concern in creating this transformation was the selective oxidation of the 9α-OH A mild oxidizing agent, such as pyridinium dichromate (PDC), was used to oxidize 13- acetyl-9-dihydrobaccatin III. Using these reaction conditions, the 7β-OH was preferentially oxidized over the 9α-OH and the oxetane ring was opened affording 13-acetyl-9-dihydro-D- seco-5, 6-dehydrobaccatin III, 3, (Scheme 1) Jones' oxidation of the 9-dihydro-13- acetylbaccatin III, 1, afforded 13-acetyl-7-oxobaccatin III, 4, wherein both the 9 -OH and the 7β-OH are oxidized simultaneously Similar to the investigation, of the oxidation products of paclitaxel, realized by Kingston (J. Org. Chem., 1998, 51, 797), 13-acetyl-7-oxobaccatin III is unstable and converts readily to the D-seco derivative, 13-acetyl-D-seco-5,6- dehydrobaccatin III, 5 When the reaction mixture, after oxidation, was quenched under non- aqueous conditions with KHCO₃, dried over MgSO , and filtered through silica gel with ether, very little conversion to the D-seco derivative resulted and the desired 13-acetyl-7- oxobaccatin III was obtained in 69% yield Alternatively, if

5 R = OAc 5a R = OH

Scheme 1

the reaction mixture, after oxidation, was diluted with EtOAc, washed with aqueous NaHCO₃, then brine, dried over MgSO₄ , filtered, evaporated and the product purified on silica gel with EtOAc, then a 100% conversion to the D-seco derivative, 5, was observed

However, 13-acetyl-7-oxobaccatin III was stable when the purification step on silica gel was omitted, despite the aqueous work-up.

The reduction of 13-acetyl-7-oxobaccatin III, 4, (Scheme 2) was then investigated. By taking advantage of the greater reactivity of the 7-oxo group, 4 was reduced using NaBFL in methanol at 4 °C. As a result, 13-acetyl-7-ep/baccatin III, 13, was produced exclusively in 82%o yield. The NOESY data show a correlation between the C-7 proton and the methyl group at C-19, and a correlation between the 7-OH and the proton at C-3, thereby confirming a C-7α-OH. This result was undesired since the original 7β-OH confirmation was expected. Kingston et al. (J. Org. Chem., 58: 3798, 1993) were able to successfully convert 1-epi-taxol to taxol™ via its 7-(S-methylthiocarboxy)taxol derivative. A more direct route was used in the present invention which employed the equilibrium reaction between the two epimers at C- 7 under basic conditions, which takes advantage of the equilibrium reaction previously studied by Fang et αl. (Synthetic Comm., 27: 2305, 1997). A similar technique had been found to be useful in the preparation of 6α-hydroxypaclitaxel, as referenced in Tetrahedron Eett., 1998, 39, 4961. Treatment of compound 13 in toluene with the bulky base 1,8- diazabicyclo[5,4,0]undec-7-ene at 80°C, yielded a single product, the 7β-OH, 14, in 29% yield along with recovered starting material in 57% yield. Although the reaction is not quantitative, the yield based on recovered starting material was 67% which is quite acceptable. According to Fang et al. (Synthetic Comm., 1997, 27: 2305), 7β-epimerization is enhanced when the 13-OAc has been cleaved. In an attempt to improve the yield of the 7α to the 7β-epimerization, the hydrolysis of the 13-OAc of 13 was performed prior to epimerization step. When 13 was treated successively with NaBHj in the THF/0.05M phosphate buffer, pH 7.0, 2/1 at 23 °C, two compounds were obtained, neither of which were identified as the expected 7-epibaccatin III. The major compound was identified as the hydrolysis of the 4-OAc from 13, which was then hydrolyzed at the C-13 position to afford the minor compound. Therefore, the final step in the synthesis was the hydrolysis of the C-13 acetyl. Compound 14 was reductively cleaved with NaBFL_t in THF/0.05 M potassium phosphate buffer; pH 7,2/1 at 23 °C to afford 2 in 48%yield. The NMR spectroscopic properties of 2 were identical to those for baccatin III known in the literature (In Progress in the Chemistry of Organic Natural Products, Springer- Verlag, New York, 61: 1, 1993).

14

Reagents: (a) Jones' reagent, acetone, 23 °C, 30 min; (b) NaBHj, 4 °C, 1 h, (c) DBU, toluene, 80 °C, 1.5 h; (d) NaBH₄, THF/0.05 M KP0₄ buffer, pH 7, 2.1, 23 °C.

Scheme 2

Paclitaxel derivatives are useful for their antitumor activity, particularly for the treatment of the same cancers for which paclitaxel has been shown active, including human lung tumors, melanoma, leukemia, mammary tumors, and colon cancer The taxane intermediates of the present invention can be used in the treatment of the same cancers for which paclitaxel and other taxanes have been used.

Paclitaxel has been shown to exhibit a very unique mechanism of action, in that it promotes the assembly of microtubules, but inhibits their disassembly, thereby interfering with the G2 and M phases of cell cycles and division. In vitro studies have shown that microtubules, once polymerized, in the presence of paclitaxel resist depolymerization by other agents such as CaCl or cold temperature, which normally depolymerize microtubules

A microtubule assembly study can be conducted, using compounds of the general Formula I

and Formula II, according to the in vitro procedures disclosed, for example in Parness et al, J.Cell Biol 91:479, 1981. In these studies, conditions can be established whereby a dynamic steady state exists between tubulin assembling into microtubules and the disassembly at the other end. This dynamic steady state can be measured spectrophotometrically, observing the absorbance of the solution at 350 nm. It has been shown that paclitaxel binds specifically and reversibly to the protein, stabilizing the microtubules in the polymerized form. This effect can be visualized by following the increase in absorbance of the solution at 350 nm. Cells treated with taxol™, or derivatives thereof, which exhibit chemotherapeutic activity, usually die as they are effectively blocked in mitosis.

The present invention also provides pharmaceutical compositions comprising one or more compounds of general Formula I and Formula II, as disclosed in the claims, in combination with one or more pharmaceutically acceptable, inert or physiologically active, diluents or adjuvants.

The present invention also provides pharmaceutical compositions comprising one or more compounds that can be prepared by further chemical manipulations of the compounds of general Formula I and Formula II (for example: deacetylation at C-13 and attaching an appropriate side chain at C-13), in combination with one or more pharmaceutically acceptable, inert or physiologically active, diluents or adjuvants.

The compounds of the instant invention can be freeze dried and, if desired, combined with other pharmaceutically acceptable excipients to prepare formulations for administration. These compositions may be presented in any form appropriate for the administration route envisaged. The parenteral and the intravenous route are the preferential routes for administration.

Compounds of general Formula I and Formula II, as disclosed in the claims, may be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. In addition, there is provided a pharmaceutical formulation comprising a compound of general Formula I or Formula II and a pharmaceutically acceptable carrier. One or more compounds of general Formula I and Formula II may be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants and if desired other active ingredients. The pharmaceutical compositions containing compounds of general Formula I and Formula II may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use may be prepared according to any known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate: granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium

alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia: dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethyene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example hepta- decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or «-propyl ?-hydroxy- benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.

Pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oils phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally- occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavouring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose Such formulations may also contain a demulcent, a preservative and flavouring and colouring agents The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension This suspension may be formulation according to known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides In addition, fatty acids such as oleic acid find use in the preparation of injectables The compound(s) of general Formula I and Formula II may be administered, together or separately, in the form of suppositories for rectal administration of the drug These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug Such materials are cocoa butter and polyethylene glycols

Compound(s) of general Formula I and Formula II may be administered, together or separately, parenterally in sterile medium The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle Advantageously, adjuvants such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle

The mode, dosage and schedule of administration of paclitaxel in human cancer patients has been studied extensively (see Ann Int Med 111:273 1989) For the compounds of this invention, the dose to be administered, whether a single dose, multiple dose, or a daily dose, will vary with the particular compound being used Factors to consider when deciding upon a

dose regimen include potency of the compound, route of administration, size of the recipient and the nature of the patient's condition.

The dosage to be administered is not subject to defined limits, but it will usually be an effective amount. It will usually be the equivalent, on a molar basis of the pharmacologically active free form produced from a dosage formulation upon the metabolic release of the active free drug to achieve its desired pharmacological and physiological effects.

An oncologist skilled in the art of cancer treatment will be able to ascertain, without undue experimentations, appropriate protocols for effective administration of the compounds of this present invention by referring to the earlier studies of taxol™ and its derivatives.

To assist in understanding the current invention, the following non-limiting examples are provided. The examples describe the chemical transformation of this baccatin III precursor, 9-dihyro- 13 -acetylbaccatin III, into baccatin III and baccatin III derivatives which in turn can be transformed into paclitaxel and other biologically active precursors and derivatives. The following examples should not be construed as specifically limiting the present invention, variations presently known or later developed, which would be in the understanding of one skilled in the art and considered to fall within the scope of the present invention as described herein.

EXAMPLES

General Experimental Procedures.

All the Nuclear Magnetic Resonance (NMR) spectra were obtained at room temperature with a UNITY-50 spectrometer operating at 499 83 MHz for ^XH, 125 697 MHz for ^{x 3} C and 76 75 MHz for ²H The NMR spectra were obtained on 5-10 mg dissolved in 0 3-0 4 mL of CDC1₃ The solvent was used as an external reference (77 0 ppm for ³ C and 7 25 ppm for ¹H and ²H) Chemical shifts are expressed in part per million (ppm) Coupling constants (J) are given in Hertz, where s, bs, t, d, dd, q and m indicate singlet, broad singlet, triplet, doublet, doublet of doublet, quadruplet and multiple., respectively Positive ion Fast Atom Bombardment Mass Spectra (FAB-MS) were obtained with a Vacuum Generators ZAB-HS double-focussing instrument using a xenon beam having 8 kV energy at 1 mA equivalent neutral current Flash chromatography was performed on Silica gel 60, 230-400 mesh (EM Science) Thin layer chromatography was conducted on Silica Gel 60 F₂₅₄ pre- coated TLC plates, 0 25 mm (EM Science) Analytical high performance liquid chromatography (HPLC) was performed on a Waters 600^E delivery system coupled to a PDA 996 detector Preparative and semi-preparative HPLC were carried out on a Waters Delta Prep 3000 instrument coupled to UV 486 Tunable Absorbance detector set at 227 nm (Waters, Montreal, Quebec, Canada) Analytical HPLC was performed with two Whatman partisil 10 ODS-2 analytical columns in series (4 6 x 250 mm) Semi-preparative HPLC was performed with two Whatman partisil 10 ODS-2 Mag 9 semi-preparative columns in series (9 4 x 250 mm) Preparative HPLC was performed with one partisil 10 ODS-2 MAG-20 preparative column (22 x 500 mm)

Example 1: Preparation of 13-acetyl-9-dιhydro-D-seco-5,6-dehydrobaccatιn III, 3, from 13- acetyl-9-dιhydrobaccatιn III, 1

13-Acetyl-9-dihydrobaccatin III (15 mg, 0 024 mmol) in CH₂C1₂ (3 mL) was treated with pyridinium dichromate (29 mg, 0 077 mmol) at 23 °C for 4 5 h The mixture was diluted in

CH₂C1₂ and washed with brine, dried over MgSO₄, filtered and evaporated in vacuo The residue was purified by semi-preparative HPLC (25-100% CH₃CN in H₂O, 50 min gradient at a flow rate of 3 mL/min) affording 13-acetyl-9-dihydro-D-seco-5,6-dehydrobaccatin III (2 mg, 13%, R_t = 32.97 min). HRFABMS m/∑ 651.2420 (M + Na⁺), calculated for C₃₃H₄₀Oι₂Na, 651.2418; 1H NMR (CDC1₃, 500 MHz) δ 8.10 (d, J = 7.6 Hz, 2H, H-2,6 of OBz), 7.61 (t, J = 7.3 Hz, IH, H-4 of OBz), 7.50 (t, J = 7.5 Hz, 2H, H-3,5 of OBz), 6.69 (d, J = 10.3 Hz, IH, H-5), 6.20 (d, J = 10.3 Hz, IH, H-10), 5.88 (d, J = 10.8 Hz, IH, H-6), 5.75 (br m, IH, H-13), 5.73 (br d, IH, H-2), 4.38 (d, J = 12.2 Hz, IH, H-20a), 4.37 (d, J - 12.7 Hz, IH, H-20b), 4.32 (d, J = 11.7 Hz, IH, OH-9), 4.19 (t, J = 11.2 Hz, IH, H-9), 3.42 (d, J = 5.8 Hz, IH, H-3), 3.24 (s, IH, OH-4), 2.73 (dd, J = 15.1, 3.4 Hz, IH, H-14a), 2.46 (dd, J = 15.6, 10.3 Hz, IH, H-14b), 2.16 (s, 3H, OAc), 2.10 (s, 3H, OAc), 1.84 (s, 3H, OAc), 1.72 (s, 3H, H-18), 1.63 (s, 3H, H-19), 1.58 (s, 3H, H-17), 1.09 (s, 3H, H-16); ¹³C NMR (CDC1₃, HMQC data) δ 206.1 (C-7), 170.9 (CO-OAc), 170.5 (CO-OAc), 169.9 (CO-OAc), 166.8 (CO-OBz), 152.8 (C-5), 138.1 (C-12), 136.7 (C-l l), 133.8 (C-4 of OBz), 130.1 (C-2,6 of OBz), 129.3 (C-l of OBz), 128.9 (C-3,5 of OBz), 124.6 (C-6), 77.6 (C-l), 75.2 (C-9), 74.8 (C-4), 73.3 (C- 10), 72.3 (C-2), 71.2 (C-13), 69.0 (C-20), 53.9 (C-3), 51.3 (C-8), 41.8 (C-15), 36.5 (C-14), 29.3 (C-16), 21.3 (OAc), 21.1 (OAc), 20.3 (OAc), 20.2 (C-17), 19.5 (C-19), 15.7 (C-18).

Example 2: Preparation of 13-acetyl-7-epibaccatin HI, 13, from 13-acetyl-7-oxobaccatin 111, 4

13-Acetyl-7-oxobaccatin III (46 mg; 0.073 mmol) was dissolved in methanol (10 mL) and treated with NaBFL (6 mg; 0.16 mmol) at 4°C, for 2 h. The reaction was quenched by dilution with brine. The solution was extracted with dichloromethane, and the combined organic layers were dried over MgSO₄, filtered and evaporated in vacuo. The residue was purified by chromatography on silica gel (EtOAc/hexane, 50/50) affording 13-acetyl-7- epzbaccatin III (38 mg, 82%). HRFABMS m/z 651.2420 (M + Na⁺), calculated for CsalLoOπNa, 651.2418; 1H NMR (CDC1₃, 500 MHz) δ 8.08 (dd, J = 7.8, 0.7 Hz, 2H, H-2,6 of OBz), 7.63 (t, J = 7.3 Hz, IH, H-4 of OBz), 7.50 (t, J = 7.5 Hz, 2H, H-3,5 of OBz), 6.83 (s, IH, H-10, 6.13 (br t, J = 8.0 Hz, IH, H-13), 5.75 (d, J = 7.3 Hz, IH, H-2), 4.95 (dd, J = 9.3, 3.2 Hz, IH, H-5), 4.66 (d, J = 11.7 Hz, IH, OH-7), 4.40 (d, J = 8.5 Hz, IH, H-20a), 4.35 (d, J = 8.5 Hz, IH, H-20b), 3.96 (d, J = 7.3 Hz, IH, H-3), 3.70 (ddd, J = 11.7, 4.6, 1.7 Hz, IH, H- 7), 2.41 (s, 3H, OAc), 2.36 (ddd, J = 15.9, 9.5, 2.0 Hz, IH, H-6a), 2.27 (om, IH, H-6b), 2.27 (om, 2H, H-14a b), 2.21 (s, 3H, OAc), 2.20 (s, 3H, OAc), 1.87 (d, J = 1.2 Hz, 3H, H-18), 1.65 (s, 3H, H-19), 1.19 (s, 3 H, H-16), 1.15 (s, 3H, H-17); ¹³C NMR (CDC1₃, HMQC data) δ 207.3 (C-9), 171.6 (CO-OAc), 170.2 (CO-OAc), 169.4 (CO-OAc), 167.0 (CO-OBz), 140.6 (C-12), 133.9 (C-4 of OBz), 133.0 (C-l l), 130.0 (C-2,6 of OBz), 129.3 (C-l of OBz), 128.6 (C-3, 5 of OBz), 82.5 (C-5), 82.1 (C-4), 79.0 (C-l), 78.1 (C-10), 77.5 (C-20), 75.4 (C-7), 75.2 (C-2), 69.6 (C-13), 57.5 (C-8), 42.3 (C-15), 40.2 (C-3), 35.8 (C-14), 35.2 (C-6), 25.8 (C-16), 22.3 (OAc), 20.8 (OAc), 20.8 (OAc), 20.6 (C-17), 15.9 (C-19), 15.0 (C-18).

Example 3: Preparation of 13-acetylbaccatin III, 14, from 13-acetyl-7-epibaccatin III, 13

To a solution of 13-acetyl-7-epi-baccatin III (7 mg; 0.011 mmol) in toulene (0.5 mL) was added l,8-diazabicyclo[5,4,0]undec-7-ene (55 μL; 0.37 mmol) and the mixture was stirred at 80°C for 1.5 h. The reaction mixture was then diluted in EtOAc and washed with dilute HC1, a saturated solution of NaHCO₃ and brine. The organic layer was then dried over MgSO₄, filtered and evaporated in vacuo. The residue was purified by semi-preparative HPLC (25- 100%.) CH₃CN in H₂O, 50 min. gradient at a flow rate of 3 mL/min) affording 13- acetylbaccatin III (2 mg, 29%, Rt = 33.58 min) and recovered 13-acetyl-7-epi-baccatin III (4 mg, 57%, R_t = 37.98 min). HRFABMS m/z 651.2420 (M + Na⁺), calculated for C_{3 3}H_{4 0} O _{1 2}Na, 651.2418; 1H NMR (CDC1₃, 500 MHz) δ 8.07 (d, J = 7.6 Hz, 2H, H-2,6 of OBz), 7.61 (t, J = 7.5 Hz, IH, H-4 of OBz), 7.48 (t, J = 7.4 Hz, 2H, H-3,5 of OBz), 6.30 (s, IH, H-10), 6.18 (dd, J = 9.5, 8.2 Hz, IH, H-13), 5.66 (d, J = 7.2 Hz, IH, H-2), 4.97 (dd, J = 9.8, 1.6 Hz, IH, H-5), 4.43 (br m, IH, H-7), 4.30 (d, J = 8.1 Hz, IH, H-20a), 4.16 (d, J = 8.5 Hz, IH, H-20b), 3.82 (d, J = 6.9 Hz, IH, H-3), 2.55 (ddd, J = 14.7, 9.4, 6.2 Hz, IH, H-6a), 2.46 (br d, J = 3.0 Hz, IH, OH-7), 2.32 (s, 3H, OAc), 2.27 (om., IH, H-6b), 2.24 (om, 2H, H- 14a/b), 2.24 (s, H, OAc), 2.20 (s, 3H, OAc), 1.90 (d, J = 1.1 Hz, 3H, H-18), 1.67 (s, 3H, H- 19), 1.23 (s 3H, H-16), 1.13 (s, H, H-17); ¹³C NMR (CDC1₃, HMQC data) δ 203.4 (C-9), 170.9 (CO-OAc), 169.6 (CO-OAc), 169.2 (CO-OAc), 166.6 (CO-OBz), 142.5 (C-12), 134.2 (C-4 of OBz), 132.2 (C-l l), 130.3 (C-2,6 of OBz), 129.3 (C-l of OBz), 128.8 (C-3, 5 of OBz), 84.6 (C-5), 80.5 (C-4), 78.7 (C-l), 76.3 (C-20), 75.8 (C-10), 75.1 (C-2), 72.4 (C-7), 69.7 (C-13), 58.1 (C-8), 45.6 (C-3), 42.6 (C-15), 35.6 (C-14), 35.6 (C-6), 26.7 (C-16), 22.4

(OAc), 21.4 (C-17), 21.1 (OAc) 20.9 (OAc) 15.0 (C-18), 9.4 (C-19). Example 4: Preparation of Baccatin III, 2, from 13-acetylbaccatin III, 14

To a solution of 13 -acetylbaccatin III (10 mg; 0.016 mmol) in THF (0.6 mL) was added 0.05 M potassium phosphate buffer, pH 7.0, (0.3 mL). The opaque solution was treated with NaBHt (2 x 5 mg; 0.13 mmol) at 23 C over a period of 10 h. The reaction was quenched by dilution with brine. The solution was extracted with dichloromethane, and the combined organic layers were dried over MgSO₄, filtered and evaporated in vacuo. The residue was purified by semi-preparative HPLC (25-100%) CH₃CN in H₂O, 50 min gradient at a flow rate of 3 mL/min) affording Baccatin III (4.5 mg, 48%, R_t = 28.45 min). The NMR spectroscopic properties of 2 were identical for those known for baccatin III.

These and other objects, as well as the nature, scope and utilization of this invention, will become readily apparent to those skilled in the art from the following description and the appended claims.

Claims

THE EMBODIMENTS IN WHICH AN EXCLUSIVE PROPERTY AND PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

A process for the preparation of Baccatin III from a compound of formula (X)

which comprises the steps of:

(i) oxidizing the hydroxy groups, on a compound of Formula X, at the C-7 and C-9 positions; (ii) reducing the resulting ketone at the C-7 position; (iii) epimerizing the resulting 7α-hydroxy; and (iv) deacylating the ester at the C-13 position.

2. A process according to claim 1, wherein said oxidizing step (i) is performed using Jones' reagent (chromium trioxide, sulphuric acid and acetone).

A process according to claim 1, wherein said reducing step (ii) is performed using NaBHj in methanol.

A process according to claim 1, wherein said epimerizing step (iii) is performed using l,8-diazabicyclo[5,4,0]undec-7-ene in toluene

5. A process according to claim 1, wherein said deacylating step (iv) is performed using NaBFL.

6. A compound of formula I

Formula I

wherein R is selected from the group comprising alpha-hydroxy and ketone.

7 A compound of Formula I according to claim 6, wherein R is alpha-hydroxy

8. A compound of Formula I according to claim 6, wherein R is ketone

A compound of Formula II

Formula II

wherein Rl is selected from the group comprising hydroxy and ketone, and R2 is selected from the group comprising hydroxy and O-acetyl.

10 A compound of Formula II according to claim 9, wherein Rl is hydroxy and R2 is O-acetyl.

11. A compound of Formula II according to claim 9, wherein Rl is ketone and R2 is O-acetyl.

12. A compound of Formula II according to claim 9, wherein Rl is ketone and R2 is hydroxy.

13. The use of the compounds of any one of claims 6 to 12 in the preparation of medicament.

14. The use according to claim 13, wherein said medicament is for use as an antitumour agent in a patient in need of such therapy. The use according to claim 14, wherein said patient has cancer

The use according to claim 15, wherein said patient is human

A composition comprising a pharmaceutically acceptable carrier and one or more of the compounds of any one of claims 6 to 12

A composition according to claim 16, formulated for administration by a convenient method chosen from the group comprising oral, topical, subcutaneous, intravenous, intramuscular, intrasternal, rectal and intranasal routes