JP2005530715A - Labeled taxane - Google Patents

Abstract

Provided is a stable labeled taxane having the feature that an isotope is present in the benzoyloxy group at the C-2 position. More particularly, one or more hydrogen or carbon atoms of the benzoyl residue at the C-2 position of the taxane skeleton are replaced with deuterium or 13C, respectively. Also provided are their preparation and their use as internal standards in analytical methods for measuring taxanes, which are effective anticancer agents.

Description

  Paclitaxel is a natural taxane diterpenoid isolated from the bark of the yew tree native to the west coast of North America (E. Baloglu, D.G.I.Kingston; J. Nat. Prod. (1999) 62: 1448-72). The structure of the compound was first elucidated and published in 1971 (MCWani, HLTaylor, MEWall, P. Coggen, ATMcPhail; J. Am. Chem. Soc. (1971) 93: 2325-7) . Because of its unique ability to inhibit the tubulin system, which is important in the process of cell mitosis (SBHorowitz, J. Fant, PBSchiff; Nature (1979) 277: 665), its dissolved in Cremophor Formulation forms are currently used for the treatment of several human cancers. Other paclitaxel analogs, referred to herein as taxanes, are under development or are already used as cytotoxic agents. The major of these taxanes is docetaxel. As a result, there is a considerable need for analytical methods to measure taxanes and their metabolites in complex biological samples such as animal and human plasma, urine, bile and tissue, in vitro cell culture media, etc. There is. Several analytical methods for measuring paclitaxel or taxane in biological fluids have been published. In any case, most of them require multi-stage sample preparation and are not very convenient for routine routine analysis. Significant improvements were obtained by using automated sample processing followed by liquid chromatography (LC) and mass spectrometry detection (MS). One important aspect of a reliable and effective analytical method is the availability of appropriate internal standards. Adding a known amount of an internal standard to an unknown sample is a known and widely used method that can compensate for the loss of the compound of interest during sample work. According to this approach, any loss of the compound of interest can be measured by the loss of the corresponding part of the internal standard. The accuracy and accuracy of this approach strongly depends on the structural similarity between the compound of interest and the internal standard. As a result, it is generally accepted that stable isotope-labeled analogs having the same molecular structure as a compound are the best internal standard for liquid chromatography mass spectrometry (LC-MS) assays. In addition, the internal standard should preferably have a molecular weight that is at least 3 mass units higher than the molecular weight of the unlabeled compound of interest. Therefore, preferably paclitaxel, docetaxel or other taxanes labeled with a large amount of stable isotope in the scaffold to improve the accuracy and specificity of the analytical method for measuring drugs or their metabolites in biological fluids Has a considerable need.

There are many chemistry related to paclitaxel and its analogs. Many papers in the literature have reported methods that can be used to introduce stable or radioactive isotopes into molecules of paclitaxel or its analogues (D. Giribone, E. Fontana, J. Labeled Compounds and
Radiopharmaceuticals, 2000, 43, p. 933-41; I. Rodriguez et al., J. Labelled Compounds and Radiopharmaceuticals, 2000, 43, p. 169-76; DDDischino et al., J. Labelled Compounds and Radiopharmaceuticals, 1995, 39, p. 173-9; S. deSuzzoni et al., J. Labelled Compounds and Radiopharmaceuticals, 1995, 36, p. 739-43; DGWalker et al., J. Labelled Compounds and Radiopharmaceuticals, 1995, 36, p. 479-91; DGWalker et al., J Labeled Compounds and Radiopharmaceuticals, 1994, 34, p. 973-80; DGIKingston et al., J. Nat. Prod., 2000, 63, p. 726-34 and WO01 / 94328-A1). These approaches involve the introduction of isotopes at one of the side chains attached to the taxane skeleton, most of which are at the C-13 position. However, these approaches require the availability of a synthon that can only be generated by a non-trivial, time-consuming, multi-step sequence of synthesis. Another possibility effective only for paclitaxel was the deuterium labeling in the 10-O-acetyl moiety described in two articles (SHHoke et al., J. Nat. Prod., 1994, 57 , p. 277-86; P. Heinstein et al., J. Chem. Soc. Perkin Trans. I, 1996, p. 845-51). The key step in both cases was the reaction of deuterated acetyl chloride with the appropriate 10-O-deacetyl paclitaxel. Again, precursors are not readily available, yields are low, and isotopic enrichment of the final compound is questionable.

  The present invention relates to stable labeled paclitaxel, docetaxel or other taxanes, their use in analytical methods and unlabeled taxane intermediates that can be readily synthesized starting from paclitaxel, docetaxel or other taxanes and commercially available advanced These preparations start from stable labeled precursors enriched in isotopes.

〔式中、
Ｒ₁は、水素原子、アセチル基またはヒドロキシ保護基を表し；
Ｒは、水素原子、ヒドロキシ保護基または式II

（式中、Ｒ₂は、ベンゾイルもしくはｔｅｒｔ−ブトキシカルボニル基を表し；Ｒ₃は、水素原子もしくはヒドロキシ保護基を表す）の残基を表し；
Ｘ、Ｘ₁、Ｘ₂、Ｘ₃およびＸ₄は、独立してＣＤ、¹³ＣＤ、¹³ＣＨまたはＣＨを表し；ＹおよびＹ₁は、ＹおよびＹ₁が両方ともＣである場合にはＸ、Ｘ₁、Ｘ₂、Ｘ₃、Ｘ₄はすべてがＣＨではないという条件で、独立してＣまたは¹³Ｃを表す〕の安定な標識タキサンを提供する。 In particular, the present invention provides compounds of formula I

[Where,
R ₁ represents a hydrogen atom, an acetyl group or a hydroxy protecting group;
R is a hydrogen atom, a hydroxy protecting group or a compound of formula II

Wherein R ₂ represents a benzoyl or tert-butoxycarbonyl group; R ₃ represents a hydrogen atom or a hydroxy protecting group;
X, X ₁ , X ₂ , X ₃ and X ₄ independently represent CD, ¹³ CD, ¹³ CH or CH; Y and Y ₁ are X when Y and Y ₁ are both C , X ₁ , X ₂ , X ₃ , X ₄ independently represent C or ¹³ C, provided that they are not all CH.

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

Preferably R ₁ represents a hydrogen atom or an acetyl group, and R represents a residue of formula II where R ₂ represents a benzoyl or tert-butoxycarbonyl group and R ₃ represents a hydrogen atom. X; X ₁ , X ₂ , X ₃ , X ₄ are ¹³ CH; Y and Y ₁ are both C.

Preferred compounds described in the present invention are:
a) 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel,
b) 2- [Ring-U- ¹³ C] -benzoyl-docetaxel, and c) 2- [Ring-U- ¹³ C] -benzoyl-10-desacetyl-baccatin III,
And more preferably has an isotopic enrichment of at least 95%, even more preferably greater than 99%.

  Another object of the present invention is the use in a method for the determination of a labeled taxane of formula I as defined above, preferably for measuring a taxane in a biological fluid.

  More preferably, the present invention provides the use of a labeled taxane of formula I as defined above as an internal standard.

Even more preferably, the present invention provides the use of 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel as an internal standard in an analytical method for measuring paclitaxel in biological fluids.

（式中、Ｒ₄はヒドロキシ保護基を表し、そしてＲ、Ｒ₁は上述の定義の通りである）の化合物を同位体標識安息香酸と反応させ、 The present invention is also a process for preparing a labeled compound of formula I comprising
i) Formula III

Wherein R ₄ represents a hydroxy protecting group and R, R ₁ are as defined above, reacted with an isotopically labeled benzoic acid;

（式中、Ｒ、Ｒ₁、Ｒ₄、Ｘ、Ｘ₁、Ｘ₂、Ｘ₃、Ｘ₄、ＹおよびＹ₁は上述の定義の通りである）の化合物からヒドロキシ保護基を除去し、式Ｉの望まれる標識タキサンを得てそして、所望する場合には、
iii）式Ｉの或る化合物を式Ｉの別の化合物に変換すること、
を含む、上記の方法を提供する。 ii) Generated formula IV

Removing the hydroxy protecting group from the compound of formula (wherein R, R ₁ , R ₄ , X, X ₁ , X ₂ , X ₃ , X ₄ , Y and Y ₁ are as defined above) To obtain the desired labeled taxane of I and, if desired,
iii) converting one compound of formula I to another compound of formula I;
The above method is provided.

Step i) uses an isotope-labeled benzoic acid (eg [ring-U- ¹³ C] benzoic acid), using a condensing agent (eg N, N′-dicyclohexyl-carbodiimide) and, if necessary, a catalyst (eg 4-pyrrole In the presence of dinopyridine).

  This reaction is carried out under an inert atmosphere, such as nitrogen, in an inert organic solvent, such as an aromatic hydrocarbon such as toluene, and at an elevated temperature, such as 60 ° C.

  The molar ratio of labeled benzoic acid and compound of formula III is 1 to 20: 1, preferably 5 to 10: 1. The molar ratio of labeled benzoic acid and condensing agent is preferably 1: 1. When present, the molar ratio of catalyst to labeled benzoic acid is about 1:15. The progress of this reaction is confirmed by analytical methods such as thin layer chromatography or high performance liquid chromatography or mass spectrometry and is usually completed within about 28 hours when no starting material of formula III is detected. At the end of the reaction, the mixture is preferably diluted with a solvent that does not dissolve the reaction by-products and filtered through, for example, a sintered glass filter funnel to remove the solid material. After evaporating the solvent to dryness, the crude material containing the compound of formula IV is recovered.

  The crude material containing the compound of formula IV is preferably purified prior to the next step ii) using techniques known to those skilled in the art. For example, preparative column chromatography using a suitable eluent, such as an organic solvent, and silica gel can be used to efficiently purify the desired compound so that the following cleavage reaction can be successfully performed.

  Step ii) is carried out by cleavage of the oxygen-protecting group bond of the compound of formula IV. Cleavage of the silicon-oxygen bond of silyl protecting the hydroxyl group can be achieved, for example, by acid treatment. On the other hand, the cleavage of the carbon-oxygen bond of the acetylated hydroxyl group can be achieved by a base, for example via a peroxidation action. Cleavage of the silicon-oxygen bond of the silyl-protected hydroxyl group is preferably under an inert atmosphere, such as nitrogen, in an anhydrous polar protic organic solvent, such as an alkanol such as methanol, and at room temperature, such as 25 ° C. To implement. The acid concentration is preferably in the range of 1-2M. The progress of this reaction is confirmed by analytical methods such as thin layer chromatography or high performance liquid chromatography or mass spectrometry and is usually completed within about 1 hour when no starting material of formula IV is detected.

  At the end of the reaction, the mixture is diluted with a water-immiscible solvent, such as ethyl acetate, then first with a base solution, such as aqueous sodium bicarbonate solution, and finally with a saturated inorganic salt, such as sodium chloride. Wash with aqueous solution.

  After drying the organic solution of the compound of interest, for example through a phase separation membrane or a sintered glass filter funnel, filtering with an inorganic desiccant such as sodium sulfate and evaporating the solvent to dryness, compound I is Collect the crude material contained.

  The crude material containing the compound of formula I is preferably purified using techniques known to those skilled in the art.

  For example, preparative column chromatography using a suitable eluent such as a mixture of water and organic solvent and C-8 reverse phase HPLC can be used to efficiently purify the desired compound. The pure compound I is recovered as an organic solvent solution by partitioning the desired HPLC eluent between a water immiscible solvent such as ethyl acetate and a saturated aqueous solution of an inorganic salt such as sodium chloride.

  After evaporating the solvent to dryness, the pure compound of formula I is recovered as a white solid.

Compounds of formula I (R represents a hydrogen atom or a hydroxy protecting group) or labeled baccatin derivatives are known to compounds of formula I (R represents a residue of formula II as defined above). It can be easily converted by a technique. In particular, the inventor refers to the method described in WO97 / 42167-A. The starting compounds of formula III (R, R ₁ and R ₄ are as defined above) can be readily obtained by techniques known to those skilled in the art summarized in the following schemes.

（式中、Ｒ₂はベンゾイルまたはｔｅｒｔ−ブトキシカルボニル基であり、そしてＲ₃はｔｅｒｔ−ブチルジメチルシリル基である）の残基である〕の化合物は、当業者に公知の技術によってパクリタキセルまたはドセタキセルから出発して得ることができる（例えばH.Park他, J. Med. Chem., 1996, 39, p. 2705-2709; D.G.I.Kingston 他, J. Med. Chem., 1998, 31, p. 3715-3726; G.I.Georg 他, Tetrahedron Letters, 1994, 35, p. 8931-8934を参照）。例えば、式VIの化合物のジクロロメタン溶液をメタノール中のベン
ジルトリメチルアンモニウムヒドロキシドの溶液（トリトンＢ^(R)）を用いて低温で窒素雰囲気中、厳密な制御条件下で処理することによって、選択的２−Ｏ−ベンゾイル開裂を達成できる。式III（式中、例えばＲおよびＲ₄はトリエチルシリル基であり、Ｒ₁はアセチルまたはトリエチルシリル基である）の化合物はバッカチンまたは１０−デアセチルバッカチンIIIから出発して、当業者に公知の技術によって得ることができる（例えばS-H.Chen 他, Bioorg. Med. Chem. Letters, 1998, 8, p. 2227-2230; I.Ojima 他, Bioorg. Med. Chem. Letters, 1999, 9, p. 3423-3428を参照）。 Formula III wherein, for example, R ₁ is an acetyl group, R ₄ is a triethylsilyl group, and R is a formula II

Wherein R ₂ is a residue of benzoyl or a tert-butoxycarbonyl group and R ₃ is a tert-butyldimethylsilyl group) is a paclitaxel or docetaxel by techniques known to those skilled in the art. (Eg H. Park et al., J. Med. Chem., 1996, 39, p. 2705-2709; DGIKingston et al., J. Med. Chem., 1998, 31, p. 3715- 3726; GIGeorg et al., Tetrahedron Letters, 1994, 35, p. 8931-8934). For example, by treating a dichloromethane solution of a compound of formula VI with a solution of benzyltrimethylammonium hydroxide in methanol (Triton B ^(R) ) at low temperature in a nitrogen atmosphere under strictly controlled conditions, 2 -O-benzoyl cleavage can be achieved. Compounds of formula III where R and R ₄ are for example a triethylsilyl group and R ₁ is an acetyl or triethylsilyl group are known to those skilled in the art starting from baccatin or 10-deacetylbaccatin III (Eg, SH. Chen et al., Bioorg. Med. Chem. Letters, 1998, 8, p. 2227-2230; I. Ojima et al., Bioorg. Med. Chem. Letters, 1999, 9, p. 3423-3428).

Example 1
2'-O- (tert-butyldimethylsilyl) -2- [ring-U- ¹³ C] -benzoyl-7-O- (triethylsilyl) paclitaxel [ring in anhydrous toluene (1.5 ml) at about 25 ° C To a stirred suspension of -U- ¹³ C] -benzoic acid (0.182 g, 1.48 mmol) was added N, N'-dicyclohexylcarbodiimide (0.3085 g, 1.50 mmol) and 4-pyrrolidinopyridine (15 mg). 0.1 mmol) was added under nitrogen. After stirring for 5 minutes, the compound 2′-O- (tert-butyldimethylsilyl) -2-debenzoyl-7-O- (triethylsilyl) paclitaxel (0.1445 g, 0.15 mmol, starting from paclitaxel, H. Park, M. Hepperle, TCBoge, RHHimes, GIGeorg, J. Med. Chem. (1996) 39: 2705-9 and by DGIKingston, AGChaudhary, MDChordia, M. Gharpure, AALGunatilaka, P. Giannakakou, YQJiang, CMLin, H. Hamel, BHLong, CRFairchild, KAJhonston, J. Med. Chem. (1998) 31: 3715-26) was charged into the reaction flask. The suspension was maintained at 60 ° C. under nitrogen with stirring for 28 hours. The starting taxane was then shown to be absent (C- with an eluent that was a mixture of water / acetonitrile / trifluoroacetic acid in a volume ratio of 75: 25: 0.1 to 10: 90: 0.1). The mixture was cooled to about 25 ° C., diluted with ethyl acetate (15 ml) and filtered to give a clear solution. Crude containing compound 2'-O- (tert-butyldimethylsilyl) -2- [ring-U- ¹³ C] -benzoyl-7-O- (triethylsilyl) paclitaxel after evaporation of the solvent under reduced pressure Material was recovered. The crude material was dissolved in a mixture of ethyl acetate and n-hexane (volume ratio 3: 7, 5 ml) and then a solvent prepared by a mixture of ethyl acetate and n-hexane (volume ratio 3: 7, 2.5 L) The system was chromatographed on a silica gel column (150 × inner diameter 6.0 mm) using the eluent. About 50 ml fractions were collected and the C-8 reverse phase with the eluent being a mixture of water / acetonitrile / trifluoroacetic acid in a volume ratio of 75: 25: 0.1 to 10: 90: 0.1 Confirmed by HPLC on column. The # 13-28 fractions were stored as needed and evaporated to dryness. Two batches of the title compound consisting of 77 mg and 47 mg having a purity of about 90% and 60% respectively were recovered as white solids. Purity was linear on a C-8 reverse phase column with HPLC (volume ratio of 75: 25: 0.1 to 10: 90: 0.1 water / acetonitrile / trifluoroacetic acid plus eluent) The retention time of the title compound (Rt = 17.7 minutes) was the same as that of the unlabeled reference sample as determined by a gradient and isocratic elution for 8 minutes, detection wavelength = 240 nM. The mass spectrum of the title compound was recorded using an electrospray ionization technique (ESI) that detects cations. The ESI mass spectrum showed a protonated molecular ion at m / z 1088 amu and also another unique cluster at m / z 1105 amu (M + NH ₄ ⁺ ). NMR spectra recorded at 400 MHz in CDCl ₃ showed the following signals expressed in chemical shifts (ppm): 8.27-8.38, m; 7.89-7.99, m; 7.67-7.83, m; 7.27-7.52, m 7.03-7.09, d; 6.45, s; 6.22-6.30, m; 5.72-5.77, dd; 5.69, d; 4.96, dd; 4.68, d; 4.48, dd; 4.32, d; 4.22, d; 3.85 d; 2.58, m; 2.47-2.56, m; 2.33-2.46, m; 2.17,
2.03.d; 1.87-2.02, m; 1.71, d; 1.69, s; 1.27, s; 1.22, s; 0.87-0.98, t; 0.80, s; 0.54-0.64, m; -0.02, s; -0.29, s.

Example 2
Crude 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel Compound 2′-O- (tert-butyldimethylsilyl) -2- [Ring-U- ¹³ C] -benzoyl-7- produced in Example 1 O- (Triethylsilyl) paclitaxel (73 mg, purity about 90%) was dissolved under nitrogen in a 1.5M solution of hydrogen chloride in cooled (about 4 ° C.) anhydrous methanol (4 ml). After stirring for 5 minutes, the clear solution was warmed to about 25 ° C. and stirred for an additional 60 minutes. The starting taxane was shown to be absent (C-8 reverse with eluent being a mixture of water / acetonitrile / trifluoroacetic acid in a volume ratio of 75: 25: 0.1 to 10: 90: 0.1). The reaction mixture was diluted with ethyl acetate (40 ml) and washed 4 times with a 4% strength by weight aqueous solution of sodium bicarbonate (3 ml each time). All aqueous washes were collected and extracted with ethyl acetate (6 ml). All organic phases were collected, washed 4 times with saturated aqueous sodium chloride solution (3 ml each time), filtered through a phase separation membrane and then evaporated to dryness under reduced pressure. The crude material containing the compound 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel (also referred to as [ ¹³ C6] paclitaxel, 67 mg, purity about 60%) was recovered as a white solid. Purity is linear on a C-8 reverse phase column with HPLC (volume ratio of 75: 25: 0.1 to 10: 90: 0.1 water / acetonitrile / trifluoroacetic acid plus eluent) The retention time of the title compound (Rt = 17.7 minutes) was the same as the unlabeled reference sample as determined by gradient and isocratic elution for 8 minutes, detection wavelength = 240 nM. The mass spectrum of the title compound was recorded using an electrospray ionization technique (ESI) that detects cations. The ESI mass spectrum showed protonated molecular ions at m / z 860 amu and also two other unique clusters at m / z 877 amu (M + NH ₄ ⁺ ) and 882 amu (M + Na ⁺ ).

Example 3
Crude 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel Compound 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel produced in Example 1 (45 mg, purity about 60%) was cooled (about 4 ° C.) Dissolved under nitrogen in a 1.5M solution of hydrogen chloride in anhydrous methanol (4 ml). After stirring for 5 minutes, the clear solution was warmed to about 25 ° C. and stirred for an additional 60 minutes. The starting taxane was shown to be absent (C-8 reverse with eluent being a mixture of water / acetonitrile / trifluoroacetic acid in a volume ratio of 75: 25: 0.1 to 10: 90: 0.1). The reaction mixture was diluted with ethyl acetate (40 ml) and washed 4 times with a 4% strength by weight aqueous solution of sodium bicarbonate (3 ml each time). All aqueous washes were collected and extracted with ethyl acetate (6 ml). All organic phases were collected, washed 4 times with saturated aqueous sodium chloride solution (3 ml each time), filtered through a phase separation membrane and then evaporated to dryness under reduced pressure. The crude material containing the compound ([ ¹³ C6] paclitaxel, 42 mg, purity about 45%) was recovered as a white solid. Purity is linear on a C-8 reverse phase column with HPLC (volume ratio of 75: 25: 0.1 to 10: 90: 0.1 water / acetonitrile / trifluoroacetic acid plus eluent) The retention time of the title compound (Rt = 17.7 minutes) was the same as the unlabeled reference sample as determined by gradient and isocratic elution for 8 minutes, detection wavelength = 240 nM. The mass spectrum of the title compound was recorded using an electrospray ionization technique (ESI) that detects cations. The ESI mass spectrum showed protonated molecular ions at m / z 860 amu and also two other unique clusters at m / z 877 amu (M + NH ₄ ⁺ ) and 882 amu (M + Na ⁺ ).

Example 4
Purification of the crude material containing 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel A solution having a concentration of about 7 mg / ml of the crude material produced in Example 2 or Example 3 was added to the solvent system dimethyl sulfoxide / Prepared in water / acetonitrile (volume ratio 25:25:50). 1-4 ml aliquots of this solution were injected into the following preparative high performance liquid chromatography system. Column = Symmetry Prep C8 (150 × 19 mm id, particle size 7 μm); mobile phase A = 75: 25 (volume ratio) water / acetonitrile; mobile phase B = 10: 90 (volume ratio) water / acetonitrile; elution = Linear gradient in mobile phase A 100% to mobile phase B 100% in 13 minutes, then isocratic elution in mobile phase B 100% for 8 minutes; flow rate = 20 ml / min; detection in progress = UV (wavelength: 254 nm, sampling (Minimum speed is 2 points / second); fraction collection = manual activation of the fraction collector. The UV absorbance plot during the experiment was followed in real time to identify the peak of compound 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel and collect the eluent corresponding to its pure product. . All collected fractions containing pure compound 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel were stored and then concentrated to about one-half of the original volume by rotary evaporation under reduced pressure. The solution was transferred to a separatory funnel, the same volume of saturated aqueous sodium chloride solution was added and extracted four times with ethyl acetate (6 ml each time). All organic phases were collected, filtered through a phase separation membrane and then evaporated to dryness from n-hexane. Compound 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel was obtained as a chemically pure white solid in excess of 98%. Purity is linear on a C-8 reverse phase column with HPLC (volume ratio of 75: 25: 0.1 to 10: 90: 0.1 water / acetonitrile / trifluoroacetic acid plus eluent) The retention time of the title compound (Rt = 10.2 minutes) was the same as that of the unlabeled reference sample as determined by gradient and isocratic elution for 8 minutes, detection wavelength = 240 nM. The ESI mass spectrum showed protonated molecular ions at m / z 860 amu and also two other unique clusters at m / z 877 amu (M + NH ₄ ⁺ ) and 882 amu (M + Na ⁺ ). The NMR spectrum recorded at 500 MHz in CDCl ₃ showed the following signals expressed in chemical shifts (ppm): 8.24-8.36, m; 7.94-8.04, m; 7.64-7.85, m; 7.33-7.57, m ; 6.98, d; 6.30, s; 6.22-6.28, m; 5.78-5.85, dd; 5.68-5.74, d; 4.98, d; 4.96, d; 4.82, dd; 4.39-4.47, m; 4.34, d; 4.24 , d; 3.84 d; 3.54, d; 2.53-2.64, m; 2.47, d; 2.41, s; 2.29-2.39, m; 2.27, s; 1.86-1.96, m; 1.83, d; 1.79, s; 1.71, s; 1.27, s; 1.17, s. The isotopic enrichment of 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel was greater than 99% as determined by mass spectrometry.

Claims (10)

Formula I

[Where,
R ₁ represents a hydrogen atom, an acetyl group or a hydroxy protecting group;
R is a hydrogen atom, a hydroxy protecting group or a compound of formula II

Wherein R ₂ represents a benzoyl or tert-butoxycarbonyl group; R ₃ represents a hydrogen atom or a hydroxy protecting group;
X, X ₁ , X ₂ , X ₃ and X ₄ independently represent CD, ¹³ CD, ¹³ CH or CH; Y and Y ₁ are X when Y and Y ₁ are both C , X ₁ , X ₂ , X ₃ and X ₄ independently represent C or ¹³ C, provided that they are not all CH.   The stable labeled taxane of formula I according to claim 1, wherein the hydroxy protecting group is selected from a silyl hydroxy protecting group and a 2,2,2-trichloroethoxycarbonyl group.   A stable labeled taxane of formula I according to claim 2, wherein the hydroxy protecting group is selected from trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl and thiamildimethylsilyl groups. Formula I according to claim 1
[Where,
R ₁ represents a hydrogen atom or an acetyl group, and R represents a compound of formula II

(Wherein R ₂ represents a benzoyl or tert-butoxycarbonyl group, and R ₃ represents a hydrogen atom), X, X ₁ , X ₂ , X ₃ , X ₄ are ¹³ CH Yes, and Y and Y ₁ are both C]. 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel,
2- [Ring-U- ¹³ C] -benzoyl-docetaxel, or 2- [Ring-U- ¹³ C] -benzoyl-10-desacetyl-baccatin III,
A stable labeled taxane of formula I, wherein   Use of a labeled taxane of formula I as defined in claim 1 in an analytical method.   Use of the labeled taxane according to claim 6 for measuring taxane in biological fluids.   Use of the labeled taxane according to claim 6 as an internal standard. Use of 2- [Ring-U- ¹³ C] -benzoyl-paclitaxel as an internal standard in an analytical method for measuring paclitaxel in biological fluids. A process for preparing a labeled compound of formula I as defined in claim 1, comprising
i) Formula III

Wherein R ₄ represents a hydroxy protecting group and R, R ₁ are as defined in claim 1, and is reacted with an isotopically labeled benzoic acid;
ii) Generated formula IV

Removing the hydroxy protecting group from the compound (wherein R, R ₁ , R ₄ , X, X ₁ , X ₂ , X ₃ , X ₄ , Y and Y ₁ are as defined in claim 1). To obtain the desired labeled taxane of formula I and, if desired,
iii) converting one compound of formula I to another compound of formula I;
Including the above method.