WO2007065869A1

WO2007065869A1 - Labelled docetaxel

Info

Publication number: WO2007065869A1
Application number: PCT/EP2006/069245
Authority: WO
Inventors: Erminia Fontana; Alberto Pignatti; Danilo Giribone
Original assignee: Nerviano Medical Sciences S.R.L.
Priority date: 2005-12-06
Filing date: 2006-12-04
Publication date: 2007-06-14

Abstract

There are provided deuterated docetaxel derivatives, characterized in that the isotopes are present in the tert-butoxy group. More particularly, one or more of the hydrogen atoms of the tert-butoxy group of docetaxel is replaced with deuterium (2H). A process for their preparation, their use as internal standard in analytical methods for determining docetaxel and their use in the treatment of leukemia/lymphoma and solid tumors, are also provided.

Description

TITLE OF THE INVENTION

LABELLED DOCETAXEL BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to deuterated docetaxel derivatives, to their use in analytical methods for determining docetaxel and its metabolites in complex biological samples, to their use in the treatment of leukemia/lymphoma and solid tumors and to a process for their preparation.

Discussion of the Background

Docetaxel, known as TAXOTERE ®, is a taxane derivative of paclitaxel having antitumor activity (see for example Crown J et al. "The taxanes: an update", 2000 Aug 5; 356(9228):507-8).

There is a considerable need of analytical methods to determine docetaxel derivatives and their metabolites in complex biological samples (e.g. animal and human plasma, urine, bile and tissues, in vitro cell culture media etc.). Several analytical methods have been published for the determination of docetaxel or taxanes in biological fluids. Anyway most of them are not very convenient for daily routine analysis requiring multi- steps sample preparation. A significant improvement was obtained by using automated sample handling procedure followed by liquid chromatography (LC) and mass spectrometry detection (MS). One crucial aspect of a reliable and validated analytical method is the availability of a suitable internal standard. The addition of known amount of an internal standard to the unknown sample is a well-known and widely used procedure that can compensate for losses of the compound of interest during sample workup. According to this approach, any loss of the compound of interest can be determined by the loss of an equivalent fraction of internal standard. The precision and accuracy of this approach is strongly dependent on the structural similarity between the compound of interest and the internal standard. As a consequence it is generally agreed that the stable isotopically labelled analogues with the same molecular structure of a compound are the best internal standards for liquid chromatography-mass spectrometry (LC-MS) assay. In addition the internal standard should have preferably a molecular weight at least three mass units higher than that of the non-labelled compound of interest. Therefore there is a considerable need of docetaxel labelled with a high amount of stable isotopes in the skeleton in order to improve the accuracy and specificity of the analytical method to determine the drug or its metabolites, preferably in the biological fluids. The chemistry of docetaxel is not trivial; in the literature, many papers reported methods that can be used to introduce stable- or radio-isotopes in the molecule of docetaxel which involve the introduction of the isotopes in one of the side chains attached at taxane skeleton, mostly in that at the C-13 position. These methods require the availability of synthons that can be prepared only according to non-trivial, time- consuming, multi-step synthetic sequences. In the Journal of Labelled Compounds & Radiopharmaceuticals (2004), 47(11), 763-777, van Tilburg, E. W. et al. describe the radiosynthesis of [^uC]docetaxel, with the ¹¹C isotope in the ferf-butoxycarbonyl (Boc) moiety by means of [^uC]ferf-butoxycarbonylation of the free amine of docetaxel.

WO 03/080587 describes and claims inter alia 2-[ring-U-¹³C]-benzoyl-docetaxel and its preparation by reacting a protected baccatin intermediate with an isotopically labelled benzoic acid.

It is well known that the C- H bond is more stable than the C-H bond. As a consequence, the deuterated forms of a drug have sometimes different properties due to various reasons such as changes in drug metabolism and pharmacokinetics, different transport processes, altered physicochemical characteristics (see for example D. J. Kushner et al, Canadian Journal of Physiology and Pharmacology (1999), 77 (2), 79- 88). In the prior art, no details are described regarding deuterated derivatives preparation for improving the profile of docetaxel molecule. Therefore, another object of the present invention is to provide a deuterated docetaxel derivative with an improved pharmacological profile.

DETAILED DESCRIPTION

This invention relates to deuterated docetaxel derivatives, to their use in analytical methods, to their use in the treatment of leukemia/lymphoma and solid tumors and to a process for their preparation.

In particular, the present invention provides a deuterated docetaxel derivative of the formula I:

wherein R_a, Rb, Rc, Ra, Re, Rf, R_g, Rh and Ri, each independently represents hydrogen or

Deleted: R represents hydrogen deuterium atom (²H)₅, and at least one of R_a, Rb, Rc, Rd, Re, Rf, Rg, Rh and R₁ represents atom or a hydroxy protecting a deuterium atom.

Preferably, the deuterated docetaxel derivatives of the formula I have at least three

deuterium atoms in the fert-butyl group, i.e. at least three of R_a, Rb, Rc, Rd, Re, Rf, Rg,

Rh and R₁ represent deuterium atoms.

The preferred compound according to the present invention is [tert-butyl-²Hg]docetaxel,

that is docetaxel having all the nine hydrogen atoms of the terf-butyl group replaced

with nine deuterium atoms of the formula Ia:

Another object of the present invention is the use of a deuterated docetaxel derivative of

the formula I as defined above in an analytical method, preferably in order to determine

a docetaxel derivative in the biological fluids.

More preferably, the present invention provides the use of a deuterated docetaxel

derivative of the formula I as defined above as an internal standard.

Still more preferably, the present invention provides the use of [tert-butyl- Hgjdocetaxel

as an internal standard in an analytical method for determining docetaxel in the

biological fluids.

The subject invention also includes a method of treatment of cancer or cell proliferative disorders which comprises administering to a mammal in need thereof an effective amount of a deuterated docetaxel derivative of the formula I as defined above, more preferably [ferf-butyl- Hc>]docetaxel.

In another embodiment, the present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of deuterated docetaxel derivative of the formula I as defined above in association with at least one pharmaceutically acceptable excipient, carrier or diluent. The present invention also provides a process for preparing a deuterated docetaxel derivative of the formula I, which process comprises reacting a compound of the formula II:

II

wherein R is hydrogen atom or a hydroxy protecting group, with a compound of formula III:

III

wherein X represents a leaving group and R_a, Rb, R_c, Rd, Re, Rf, Rg, Rh and R₁ are as defined above and removing the hydroxy protecting group R from the resultant compound, if necessary, to obtain a compound of the formula I as defined above.

The hydroxy protecting groups are preferably selected from silyl groups, such as trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl or siamyldimethylsilyl group and 2,2,2-trichloroethoxycarbonyl group.

The leaving group which X represents is preferably a leaving group typically used to introduce a ferf-butoxycarbonyl moiety such as for example 2-(iminooxy)-2- phenylacetonitrile, O-ferf-butoxycarbonyl, O-haloCi-Cβ alkyl (such as 1,2,2,2- tetrachloroethoxy), O-haloaryl (such as pentachlorophenoxy), azide (N3), imidazole, S-(2-mercapto-4,6-dimethylpyrimidine), or O-p-nitrophenyl group.

The reaction is performed with a deuterium labelled ferf-butoxycarbonyl group derivative of formula III as defined above in an appropriate solvent, optionally in the presence of a condensing agent or a catalyst. Preferably, X represents 2-(iminooxy)-2- phenylacetonitrile group and the resultant derivative of formula III is 2-(tert- [²H₉]butoxycarbonyloxyimino)-2-phenylacetonitrile of formula

(C²H₃)₃CO-C(O)-O-N=C(C₆H₅)-CN.

Preferably, the reaction is carried out at room temperature, for example 22°C. The progress of this reaction is checked by an analytical method, for example thin layer chromatography, high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS), and is complete when no starting material of formula II is detected, generally within about 18 hours. The resultant crude material containing the compounds of formula I is preferably purified by using techniques well known in the art.

For example, preparative column chromatography using C-18 reverse phase HPLC along with appropriate eluants as mixtures of water and organic solvents may be used to effectively purify the desired compound. The pure compound I is recovered as an organic solvent solution by partitioning the HPLC eluate of interest between a solvent non-miscible with water, for example ethyl acetate, and a saturated water solution of an inorganic salt, for example sodium chloride.

The pure the compounds of formula I are recovered as a white solid after solvent evaporation to dryness.

The removal of the hydroxy protecting group which R may represent can be easily carried out by well known procedures for obtaining a compound of formula I. The starting compounds of formula II are known and can be easily obtained according to techniques well known in the art. For example, one method of preparation is shown in the the following scheme, wherein R is as defined above, tBu is ferf-butyl group, TFA is trifluoroacetic acid and DCM is dichloromethane:

IV Il

The starting compound of formula IV in which R is a hydrogen atom is docetaxel; the compounds wherein R is a ferf-butyldimethylsilyl group can be obtained starting from docetaxel according to techniques well known in the art. Alternatively, a compound of formula II wherein R is hydrogen atom can be obtained as described in Journal of Medicinal Chemistry, (1991), 34(3), 992-8, by Gueritte-Voegelein, Francoise et al. The deuterium presence affords certain therapeutic advantages resulting, for example, from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or use of alternative formulations and, hence, the compounds of formula I are preferred to the docetaxel.

The compounds of formula I are useful for the same cancers for which docetaxel has been shown active, including human ovarian tumors, mammary tumors, and malignant melanoma, lung tumors, gastric tumors, colon tumors, head and neck tumors, and leukemia. See, e. g., the clinical pharmacology of docetaxel is reviewed by Earhart, Robert H. in Seminars in Oncology (1999), 26(5, Suppl. 17), 8-13.

Docetaxel is extensively metabolized and the major human metabolites originated from the oxidation of the ferf-butyl moiety in the C-13 side chain (see for example A. Sparreboom et al., Drug Metabolism and Disposition (1996), 24 (6), 655-658).

The replacement of hydrogen with deuterium in the ferf-butyl moiety can alter and significantly improve the pharmacological profile of docetaxel. The biological activity, the physicochemical, toxicological an pharmacokinetic properties of the compounds of formula I of the invention can be confirmed using standard chemical and biochemical assays, modeling techniques and appropriate pharmacokinetic and/or pharmacodynamic models. For example, the improvement of metabolic stability of the compounds of formula I of the invention can be confirmed using well-known procedures. The compounds of the invention can be formulated per se in pharmaceutical preparations. The deuterated docetaxel derivatives of the invention can be utilized in the treatment of cancers, due to their cytotoxic, antitumor activity. The new compounds are administrable in the form of tablets, pills, powder mixtures, capsules, injectables, solutions, suppositories, emulsions, dispersions, food premix, and in other suitable form. The pharmaceutical preparation which contains the compound is conveniently mixed with a non-toxic pharmaceutical organic carrier or a non-toxic pharmaceutical inorganic carrier, usually about 0.01 mg up to 2500 mg, or higher per dosage unit, preferably 50-500 mg. Typical of pharmaceutically acceptable carriers are, for example, mannitol, urea, dextrans, lactose, potato and maize starches, magnesium stearate, talc, vegetable oils, polyalkylene glycols, ethyl cellulose, poly(vinylpyrrolidone), calcium carbonate, ethyl oleate, isopropyl myristate, benzyl benzoate, sodium carbonate, gelatin, potassium carbonate, silicic acid, and other conventionally employed acceptable carriers. The pharmaceutical preparation may also contain non-toxic auxiliary substances such as emulsifying, preserving, wetting agents, and the like as for example, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene monostearate, glyceryl tripalmitate, dioctyl sodium sulfosuccinate, and the like.

Exemplary of a typical method for preparing a tablet containing the active agents is to first mix the agent with a non-toxic binder such as gelatin, acacia mucilage, ethyl cellulose, or the like. The mixing is suitably carried out in a standard V-blender and usually under anhydrous conditions. Next, the just prepared mixture can be slugged through conventional tablet machines and the slugs fabricated into tablets. The freshly prepared tablets can be coated, or they can be left uncoated. Representative of suitable coatings are the non-toxic coatings including shellac, methylcellulose, carnauba wax, styrene-maleic acid copolymers, and the like. For oral administration, compressed tablets containing 0.01 milligram, 5 milligrams, 25 milligrams, 50 milligrams, 500 milligrams, etc., up to 2500 milligrams are manufactured in the light of the above disclosure and by art known fabrication techniques well known to the art and set forth in Remington's Pharmaceutical Science, Chapter 39, Mack Publishing Co., 1965.

To formulate the tablet, the active compound, cornstarch, lactose, dicalcium phosphate and calcium carbonate are uniformly blended under dry conditions in a conventional V- blender until all the ingredients are uniformly mixed together. Next, the cornstarch paste is prepared as a 10% paste and it is blended with the just prepared mixture until a uniform mixture is obtained. The mixture is then passed through a standard light mesh screen, dried in an anhydrous atmosphere and then blended with calcium stearate, and compressed into tablets, and coated if desired. Other tablets containing 10, 50, 100, 150 mgs, etc., are prepared in a like fashion.

The following formulation A is an example of a tablet formulation comprising a compound of the invention.

FORMULATION A

Ingredients: Per tablet, mg.

Active compound 50.0

Cornstarch 15.0

Cornstarch paste 4.5

Calcium carbonate 15.0

Lactose 67.0

Calcium stearate 2.0

Dicalcium phosphate 50.0

The manufacture of capsules containing 10 milligrams to 2500 milligrams for oral use consists essentially of mixing the active compound with a non-toxic carrier and enclosing the mixture in a polymeric sheath, usually gelatin or the like. The capsules can be in the art known soft form of a capsule made by enclosing the compound in intimate dispersion within an edible, compatible carrier, or the capsule can be a hard capsule consisting essentially of the novel compound mixed with a non-toxic solid such as talc, calcium stearate, calcium carbonate, or the like. Capsules containing 25 mg, 75 mg, 125 mg, and the like, of the novel compound, singularly or mixtures of two or more of the novel compounds are prepared, for example, as follows:

FORMULATION B

Ingredients Per Capsule, mg.

Active compound 50.0

Calcium carbonate 100.0

Lactose, U.S.P. 200.0

Starchl 30.0

Magnesium stearate 4.5 The above ingredients are blended together in a standard blender and then discharged into commercially available capsules. When higher concentrations of the active agent are used, a corresponding reduction is made in the amount of lactose.

The compounds of the invention can also be freeze dried and, if desired, combined with other pharmaceutically acceptable excipients to prepare formulations suitable for parenteral, injectable administration. For such administration, the formulation can be reconstituted in water (normal, saline), or a mixture of water and an organic solvent, such as propylene glycol, ethanol, and the like. The dose administered, whether a single dose, multiple dose, or a daily dose, will of course, vary with the particular compound of the invention employed because of the varying potency of the compound, the chosen route of administration, the size of the recipient and the nature of the patient's condition. The dosage administered is not subject to definite bounds, but it will usually be an effective amount, or the equivalent on a molar basis of the pharmacologically active free form produced from a dosage formulation upon the metabolic release of the active drug to achieve its desired pharmacological and physiological effects.

Typically the compounds of the invention can be administered by intravenous injection at doses of 1-500 mg per patient per course of treatment, preferable with doses of 2-100 mg, the exact dosage being dependent on the age, weight, and condition of the patient. An example of a suitable formulation for injection is using a solution of the compound of the invention in a mixture of polysorbate alcohol and dehydrated alcohol (e.g. , 1 : 1) followed by dilution with 5% dextrose in water prior to infusion or injection.

EXAMPLE 1

10-Desacetyl-N-debenzoyl paclitaxel (IL R=H)

Docetaxel (IV, R=H; 199.8 mg; 0.247 mmol) in dichloromethane (3 ml) was stirred at 0° C for 20 minutes then trifluoroacetic acid (1 ml) was dripped into the reaction flask. The reaction mixture was stirred for 75 minutes at 0⁰C. At the end of reaction [checked by the following HPLC method: Xterra MS Cl 8 column (mm 100 x 4.6 ID, 5μm, by Waters) eluting with water (A) and acetonitrile (B): from 90%A to 0%A in 15 minutes; flow rate: 1 ml/min; column temperature: 40⁰C; analytical wavelength: 254 nm] a NaHCθ3 solution (8% w/v) was added at 0⁰C up to pH 8. The aqueous layer was transferred into a separating funnel and extracted with ethyl acetate (3 x 10 ml). The organic phases were combined, dried (Na2SO4) and evaporated to dryness. A crude material was recovered (194.5 mg) 67% chemically pure which was purified by flash- chromatography on a silica gel cartridge using a mixture of ethyl acetate: methanol 95:5 by volume as eluting solvent system. After combining the fractions as appropriate and solvent evaporation to dryness, the title compound was obtained as a white solid (150.7 mg; 0.213 mmol) 86% chemically pure (determined by HPLC method described before; Rt = 8.1 minutes), with a yield of about 85%.

EXAMPLE 2

10-Desacetyl-N-debenzoyl paclitaxel prepared in example 1 (150.7 mg; 0.213 mmol) and 2-(ferf-[²H₉]butoxycarbonyloxyimino)-2-phenylacetonitrile (60.9 mg; 0.238 mmol) were dissolved in pyridine (5 ml). The reaction mixture was stirred at room temperature for 18 hours. At the end of reaction (checked by HPLC method described before) the reaction mixture was evaporated to dryness and the crude title compound was obtained as a yellow oil (315.8 mg) which was purified by preparative HPLC [Xterra MS Cl 8 column (100 x 30 mm ID, 5μm, by Waters) eluting with water: acetonitrile 95:5 by volume (A) and water: acetonitrile 5:95 by volume (B): from 100%A to 0%A in 30 minutes; flow rate: 43 ml/min; column temperature: ambient; analytical wavelength: 254 nm]. The real time UV -profile plot of each run was followed visually to identify the peak of the title compound. The collected fractions were combined as appropriate. After evaporation of most of the acetonitrile, the obtained solution was transferred into a separating funnel and extracted with ethyl acetate (4 x 50 ml). All the organic extracts were pooled and dried (Na2SO4). After solvent evaporation under vacuum, the title compound (105.0 mg; 0.128 mmol) was recovered as a white solid with a yield of approximately 60%. The chemical purity was found to be > 97% (determined by HPLC method described before) and the retention time of the title compound (Rt = 11.0 minutes) was the same of an authentic non-labelled sample. The mass spectrum of the title compound was recorded using the electrospray ionization technique (ESI) with positive ion detection. The ESI mass spectrum showed the protonated molecular ion ([M+H]⁺) at m/z 817 amu. The NMR spectrum recorded in DMSO-d6 at 500 MHz showed the following signals expressed as chemical shifts (ppm): 8.00-8.04, d; 7.73- 7.78, t; 7.64-7.69, t; 7.39-7.46, m; 7.32-7.37, m; 7.22-7.27, t; 5.87-5.94, m; 5.44-5.48 d; 5.03-5.07, d; 4.97-4.99, d; 4.914.97, m; 4.54, s; 4.36-4.41, t; 4.03-4.13, m; 3.69-3.74, d; 2.28-2.37, m; 2.26, s; 1.9-2.0, m; 1.79, s; 1.67-1.83, m; 1.57, s; 1.06, s; 1.03, s.

Improvement of metabolic stability

Comparison of the metabolic stability in human CYP3A4 enzyme of [ferf-butyl- Hgjdocetaxel prepared in example 2 with docetaxel itself in an in vitro metabolic stability assay indicated that after 1 hour of incubation at 37°C of a 2 μM solution, the percentage of the original drug remaining for [ferf-butyl- Hc_>]docetaxel was 90% and that for docetaxel was 65%.

Claims

l A deuterated docetaxel denvative of the formula I:

I

wherein R_a, Rb, R_c, Rd, Re, Rf, Rg, Rh and R₁, each independently represents hydrogen or

Deleted: R represents hydrogen deuterium atom, and at least one of R_a, Rb, R_c, Rd, Re, Rf, R_g, Rh and R₁ represents a atom or a hydroxy protecting deuterium atom.

2. A deuterated docetaxel derivative according to claim 1 having at least three

deuterium atoms in the tert-butyl group, i.e. wherein at least three atoms of R_a, Rb, R_c,

Rd, Re, Rf, Rg, Rh and R₁ represent deuterium atoms.

3. A deuterated docetaxel derivative according to claim 1 or 2 which is

[ter?-butyl-²H₉]docetaxel.

4. A deuterated docetaxel derivative according to any of preceding claims of the

formula Ia:

5. Use of a deuterated docetaxel derivative as defined in claim 1 in an analytical

method.

6. Use of a deuterated docetaxel derivative according to claim 5 in order to determine a

Deleted:

taxanejn the biological fluids.

7. Use of a deuterated docetaxel derivative according to claim 5 as an internal standard.

8. Use of [ferf-butyl- Hc_>]docetaxel as an internal standard in an analytical method for

Deleted: determining docetaxel jn the biological fluids.

9. A pharmaceutical composition comprising a deuterated docetaxel derivative as

defined in claim 1 and a pharmaceutically acceptable carrier.

10. A pharmaceutical composition according to claim 9 wherein the deuterated

docetaxel derivative is [ferf-butyl-²H₉]docetaxel.

11. The pharmaceutical composition of claim 9 or 10 that is in tablet form.

12. A process for preparing a deuterated docetaxel derivative as defined in claim 1,

which process comprises reacting a compound of the formula II:

II

wherein R is hydrogen atom or a hydroxy protecting group, with a compound of

formula III:

III

wherein X represents a leaving group and R_a, Rb, R_c, Rd, Re, Rf, R_g, Rh and R₁ are as

defined in claim 1 and removing the hydroxy protecting group R from the resultant

compound, if necessary, to obtain a compound of the formula I as defined above.

13. A process according to claim 12 wherein R is hydrogen atom and X is 2- (iminooxy)-2 -phenylacetonitrile.