DD296485A5 - PROCESS FOR PREPARING TAXOL DERIVATIVES - Google Patents

Abstract

There are described processes for the preparation of taxol derivatives of the formula (II) and the salts thereof, in which R and R are each H or the radical of an amino acid from the group alanine, leucine, isoleucine, valine, phenylalanine, proline, lysine and arginine or a Group of formula (III), wherein n is an integer having a value of 1 to 3 and R2 and R3 are each hydrogen atoms or an alkyl group having 1 to 3 carbon atoms, or wherein R2 and R3 together with the nitrogen atom to which they form a saturated heterocyclic ring having 4 or 5 carbon atoms, with the proviso that at least one of R and R1 is not hydrogen. Also described are pharmaceutical compositions containing such derivatives. The Taxolderivate have increased Wasserloeslichkeit compared to taxol and have an excellent anti-tumor activity. Formulas (II) and (III) {taxol; taxol derivatives; pharmaceutical compositions; water solubility; Antitumoraktivitaet}

Description

OH

C = O

R-

in which R ² , R ³ and η are as defined above, whereby a 2'-

protected, 7-substituted taxol receives; and (c) the protecting group is removed at the 2-position.

13. The method according to claim 12, characterized in that one carries out the removal of the protective group by reacting the 2'-protected, 7-substituted taxol with a mixture of zinc and acetic acid.

Field of application of the invention

The application of the present invention is in the field of drugs for combating tumors.

Characteristic of the known state of the art

Taxol is a known diterpenoid sesquiterpene originally isolated from bark of Taxus brevifolia and has the structure:

It has been shown that taxol has potent antitumor activity against a variety of tumors. However, this compound is only slightly soluble in water, so that considerable difficulties in the development of suitable

Medicinal preparations suitable for human therapy have occurred. Some preparations of Taxol for injection or intravenous infusion have been developed using predominantly Cremophore EL® as the excipient to overcome the problems with the low water solubility of Taxol. However, Cremophore itself has some toxicity and causes idiosyncratic histamine release and anaphylactoid-like reaction, so that the use of this carrier is not a desirable way of solving the problems involved in the development of favorable preparations of taxol.

Object of the invention

The aim of the invention is to provide new drugs for combating tumors.

Explanation of the essence of the invention

It is an object of the present invention to provide novel taxol derivatives which have better water solubility than taxol but which possess the same or similar beneficial antitumor activity and cytotoxic activity as taxol. It is also desirable to provide preparations of taxol derivatives which utilize non-toxic excipients, thereby avoiding the use of toxic carriers, such as creamophores. Finally, taxol derivatives are to be provided which have good stability at pHs which are suitable for the preparation of pharmaceutical preparations (pH 3 to 4), but which rapidly degrade in vivo at physiological pH values (pH 7.4) so that they act as drug precursors for taxol

 The invention relates to a process for the preparation of taxol derivatives of the general formula II and the salts thereof

O

OR

in the R and R 'are each H or a group of the formula

C = O

(? ^H 2> n

where η is an integer having a value of 1 to 3 and R ² and R ^{3 are} each hydrogen or an alkyl group having 1 to 3 carbon atoms, or wherein R ² and R ³ together with the nitrogen atom they are bonded, one form saturated heterocyclic ring having 4 or 5 carbon atoms, with the proviso that at least one of the radicals R and R ^{1 is} not a hydrogen atom, which is characterized in that taxol with a compound of formula IV

OH

C = O

(IV)

RR ^:

in which η, R ² and R ^{3 have} the meaning defined above, reacted.

Furthermore, the invention relates to a process for the preparation of taxol derivatives of the formula II and the salts thereof

O

OR

in which R and R 'each represent hydrogen atoms or the radical of an amino acid, with the proviso that at least one of the radicals R and R' does not represent a hydrogen atom, characterized in that one converts taxol with an N-protected amino acid and then the protective group of the amino acid removed

 Furthermore, the invention relates to a process for the preparation of taxol derivatives of the general formula II and the salts thereof

O

OH

in which R 'is the radical of an amino acid from the group alanine, leucine, isoleucine, valine, phenylalanine, proline, lysine and arginine or a radical of the formula III

C = O

R °

in which η is an integer with a value of 1 to 3 and R ² and R ^{3 are} each hydrogen or an alkyl radical having 1 to 3 carbon atoms or in which R ² and R ³ together with the nitrogen atom to which they are attached, form a saturated heterocyclic ring of 4 or 5 carbon atoms which is characterized by reacting (a) taxol with a hydroxyl-protecting group compound to form 2'-protected taxol;

(b) reacting the 2'-protected taxol with alanine, leucine, isoleucine, valine, phenylalanine, proline, lysine, arginine or a compound of the following formula

C = O

R ² \ i

wherein R ² , R ³ and η are as defined above to give a 2'-protected, 7-substituted taxol; and

(c) remove the protecting group in the 2-position.

In the above formula III, when R ² and / or R ^{3 have} a meaning other than hydrogen atoms, these compounds can be regarded as "alkylated amino acids" and hence are sometimes referred to as such below.

Thus, the compounds of formula II are generally considered to be 2'- and / or 7-esters of taxol with amino acids or alkylated amino acids.

The invention thus comprises (a) derivatives derivatized at the 2'-hydroxyl group of taxol, (b) derivatives esterified with taxol at the 7-hydroxyl group and (c) derivatives esterified at the 2'- and 7-hydroxyl groups.

Among the compounds of general formula II above, the following specific compounds are preferred embodiments of the invention.

1. 2 '- (N, N-diethylaminopropionyl) taxol

2. 2 '- (N, N-dimethyl glycol) taxol

3. 7- (N, N-dimethylglycyl) taxol

4. 2 '-, 7-Di- (N, N-dimethylglycyl) -taxol

5. 7- (N, N-diethylaminopropionyl) taxol

6. 2 ', 7-Di- (N, N-diethylaminopropionyl) taxol

7. 2 '- (L-glycyl) taxol

8. 7- (L-glycyl) taxol

9. 2 ', 7-di (L-glycyl) taxol

10. 2 '- (L-Alanyl) taxol

11. 7- (L-Alanyl) taxol

12. 2 ', 7-di (L-alanyl) taxol

13. 2 '- (L-leucyl) taxol

14. 7- (L-Leucyl) taxol

15. 2 ', 7-Di- (L-leucyl) taxol

16. 2 '- (L-isoleucyl) -taxol

17. 7- (L-isoleucyl) -taxol

18. 2 ', 7-di (L-isoleucyl) taxol

19. 2 '- (L-Valyl) -taxol

20. 7- (L-Valyl) taxol

21. 2 ', 7-di (L-valyl) taxol

22. 2 '- (L-phenylalanyl) taxol

23. 7- (L-phenylalanyl) taxol

24. 2 ', 7-di (L-phenylalanyl) taxol

25. 2 '- (L-prolyl) taxol

26. 7- (L-prolyl) taxol

27. 2 ', 7-di (L-prolyl) taxol

28. 2 '- (L-lysyl) -taxol

29. 7- (L-lysyl) taxol

30. 2 ', 7-di (L-lysyl) taxol

31. 2 '- (L-Glutamyl) -taxol

32. 7- (L-Glutamyl) taxol

33. 2 ', 7-Di- (L-glutamyl) -taxol

34. 2 '- (L-Arginyl) -taxol

35. 7- (L-Arginyl) taxol

36. 2 ', 7-di- (L-arginyl) taxol.

Production Process According to the invention, it has been found that of the chemically reactive hydroxyl groups of taxol in the 2'- and 7-positions, especially the 2'-hydroxyl group is chemically more reactive than the 7-hydroxyl group. This observation is exploited according to the invention to direct the position of the derivatives in the production process.

A. Preparation of 2'-ester derivatives

The 2'-ester derivatives can be prepared by one of two methods, depending on whether it is derivative formation with an amino acid or with an alkylated amino acid.

For the preparation of esters of alkylated amino acids, the following reaction scheme is followed:

Scheme I

Alkylated amino acid + taxol -> 2'-ester-taxol derivative

OH I C = O

(CH.J

2 η

(I)

O-

wherein n, R ² and R ^{3 have} the meaning defined above.

For the preparation of esters of amino acids, the following reaction scheme follows:

Scheme II

N-protected amino acid + taxol - > 2'-N-protected amino

acid taxol derivative

Cleavage of the protecting group

2'-Aminosäureestertaxolderivat

In both of the above reaction schemes (Schemes I and II), the reaction of the alkylated or protected amino acid is carried out in the presence of a condensing agent and with or without the additional presence of a catalyst, preferably at room temperature.

Suitable condensing agents include carbodiimides such as dicyclohexylcarbodiimide (DCC). Suitable catalysts include 4-dimethylaminopyridine (DMAP) and pyridine.

In the reaction scheme (II), various known amino acid protecting groups can be used and commercial protected amino acids can be used as starting materials. It is possible to use amino acids protected with t-BOC, FMOC or carbobenzyloxy (CBZ). Amino acids protected with t-BOC or FMOC groups are preferred. Deprotection of the t-BOC group on the 2'-esters using aqueous formic acid and other organic acids results in degradation of the product as well as stereochemical modification. The use of 99% formic acid leads to better results. In the case of FMOC-protected amino acid esters, the recovery of the product depends on the working conditions. Thus, the conditions employed in the last stage of deprotection to remove the t-BOC protecting group can cause undesirable modifications to the free hydroxyl group at the 7-position of the tolylene molecules. These undesirable modifications apparently consist of stereochemical modifications of the molecule

 The following are examples of N-protected alanine compounds:

CH ₃ OI Ii CH ₃ -COC-NH-CH-COOH

t-BOC-protected

CH ₃

CH ₃

Il

> -CHO-OC-NH-CH-COOH I CH ₃

FMOC protected.

As mentioned above, according to the present invention, it has been found that the 2'-hydroxyl group of taxol is more chemically reactive than the 7-hydroxyl group. Thus, in both of the above Schemes I and II, substitution or esterification is directed to the 2'-position when the alkylated or N-protected amino acid is reacted with taxol in a molar ratio of 1: 1 or slightly higher. Such reaction of equimolar amounts leads to the formation of a large excess of the 2'-ester-taxol derivative, although small amounts of a 7-ester-taxol derivative can also be formed as a by-product.

B. Preparation of the 7-Ester Scheme III

Since the 2'-hydroxyl group of taxol is more reactive than the 7-hydroxyl group, this esterification requires a different method than in the preparation of the 2'-derivatives. Thus, to prepare the 7-esters, a process is used in which the 2'-hydroxyl group is first protected or blocked, then the 7-hydroxyl group is esterified and then the 2'-protecting or blocking group is removed.

Taxol + protecting group reagent

2'-protected - | taxol

Deprotection in the 2-position

alkylated amino acid

2-esterified taxol < 2'-protected, (7-alkylated

Amino acid) -substituted taxol

Various blocking groups can be used to block the 2-position of taxol, such as are known in the art. Using an example of such a blocking group and using one example of an alkylated amino acid, Reaction Scheme I can be summarized as follows:

Taxol + chloroformic acid 2,2,2-trichloro-

ethyl ester (troc)

2- (troc) -taxol

7- (Ν, Ν-dimethylglycyl) -taxol

N, N-dimethyl glycine

Condensation medium

catalyst

Deprotection in the 2'-position

< 2 '- (troc) -7- (N, N-Dime)

thylglycyl) -taxol

In the above reaction scheme, the reaction of the 2'-troc taxol and the alkylated amino acid is carried out in the presence of a condensing agent and a catalyst. Suitable condensing agents and catalysts are the same products as discussed for Schemes I and II above. The deprotection of 2 '- (troc) -taxol can be carried out, for example, using a mixture of zinc and acetic acid.

Scheme IV

Alternatively, 7-substituted taxol derivatives can be prepared by a method in which taxol is first reacted with 2 to 3 equivalents of an N-blocked amino acid to produce 2 ', 7-disubstituted taxol, followed by deprotection of the amino acids in the 2'. and 7'-position is carried out and finally the amino acid is cleaved in the 2'-position. This method is shown below for t-BOC protected alanyl as an example of a blocked amino acid.

Taxol + N-t-BOC-alanine (2 equivalents)

2 ·, 7-di- (t-BOC-L-alanyl) -taxol

Amino acid protection group removal

Cleavage of 2'-alanyl from 2 ', 7-di- (L-alanyl) -taxol

7- (L-alanyl) -taxol

In this process, similar to Scheme III, the reaction of taxol with the protected amino acid is carried out in the presence of a condensing agent and a catalyst. The deprotection of the amino acids is carried out according to known methods for the removal of amino acid protecting groups, for example by mild acid treatment, such as with formic acid.

The cleavage of the 2'-amino acid is carried out by adjusting the pH of the solution of 2 ', 7- (amino acid) taxol to 7 to 7.4, for example, by mixing the 2', 7-di (amino acid ) taxols with a phosphate buffer of pH 7 to 7.4.

Adjusting the pH in this manner results in cleavage of the 2'-amino acid to produce the desired 7- (amino acid) taxol.

Thus, for example, taxol is allowed to react with 2 to 3 molar equivalents of N-protected amino acid (protected with t-BOC, CBZ or FMOC) in methylene chloride in the presence of DCC and a catalytic amount of 4-dimethylaminopyridine. In this way, the protected amino acid is introduced at the 2 'and 7 positions. The protecting groups are removed by suitable deprotecting agents (e.g., acid, mild base or hydrogenation). The 2 ', 7-bisamino acid derivative of taxol is allowed to stand at neutral pH for 24 to 48 hours in phosphate buffer to selectively deprotect in the 2'-position to give the 7-substituted derivative of taxol.

A similar reaction scheme can also be used to form 7-substituted taxol derivatives with alkylated amino acids. Such a reaction is similar to the above reaction except that the N-protected amino acid is replaced by the desired alkylated amino acid and the deprotection step is eliminated.

This scheme can be represented as follows.

Scheme V

Taxol + alkylated amino acid -> (2 equivalents)

2 ·, 7-disubstituted taxol

Cleavage of the 2'-alkylated amino acid

7-substituted taxol

C. Preparation of 2 ', 7-Disubstituted Taxol Derivatives

The disubstituted derivatives can be prepared using the above methods or parts of these methods. These methods can be described as follows:

Scheme Vl

Substitution by alkylated amino acids

Taxol + alkylated amino acid (2 to 3 equivalents)

2 ', 7- (disubstituted alkylated amino acid) taxol

Scheme VIl

Substitution by amino acids

Taxol + N-protected amino-> 2 *, 7- (disubstituted)

acid-protected amino acid) taxol

(2-3 equivalents)

Amino acid protecting groups removal

2 ', 7- (disubstituted-amino) -taxol

Scheme VI is essentially the same as Scheme I above, except that the reaction takes place with 2 equivalents of the alkylated amino acid. Further, while the process of Scheme I can be carried out in the presence or absence of a catalyst, the process of Scheme VI requires the presence of a catalyst due to the reduced chemical reactivity of the 7-hydroxyl group of taxol.

Scheme VII is basically the same as Scheme II above. However, it was noted above that in Scheme II, FMOC is the preferred protecting group to avoid steric modifications at the 7-position during deprotection. However, this difficulty does not occur in Reaction Scheme VII, since the 7-hydroxyl group is not free. Thus, various protecting groups can be used, including t-BOC and FMOC.

It is believed that one skilled in the art can, by way of description, make any compounds falling within the scope of the present invention. The following examples are merely illustrative of the methods of preparation.

embodiments

Example I

2 '- (NN-dimethylglycyl) -taxol j _{ot he} salts thereof

CR ¹

v. η;

^T CH > OCCCH ₃

1.R = O = C-CH ₂ -N (CH ₃ I ₂ , R '= H

2.R = O = 6-CH ₂ -N (CH ₃ ) ₂ .CH ₃ SO ₃ HR '= H

(a) Compound 1 - 2 '- (N, N-dimethylglycyl) taxol

A solution of taxol (0.21 g, 0.246 mmol) and Ν, Ν -dimethylglycine (0.0254 g, 0.246 mmol) in anhydrous methylene chloride (12 mL) is washed with!, S-dicyclohexylcarbodiimide (0.15 g, 0.72 mmol). and 4-dimethylaminopyridine (0.025 g, 0.2 mmol). The reaction mixture is stirred for 1 day under anhydrous conditions. Then another 50 mg of DCC are added and the stirring is continued for a further 6 hours. Then the reaction mixture is filtered and the filtrate is evaporated under nitrogen. The residue is chromatographed on silanized silica gel (35 g, 26 cm) and eluted successively with ethyl acetate: petroleum ether (1: 1) and ethyl acetate. In the ethyl acetate: petroleum ether fractions occurs during slow evaporation, a solid, which is filtered off. The mother liquor is concentrated, petroleum ether is added until turbidity occurs, and the mixture is set aside to give further product. The total yield is 0.14 g (61%). F. 168-171 ⁰ C (Zers.). In the NMR spectrum of compound 2 (300 MHz, CDCl ₃ ), the resonance of the 2'-proton is shifted from 4.71 ppm in taxol to 5.59 ppm. This is consistent with the esterification in the 2'-position. All other resonances of the spectrum are consistent with the associated structure. The HPLC purity is 98 to 99.5%. Mass spectrum: (FAB) m / e 939. (M + H) ⁺

C ₅₁ H ₆₈ N ₂ O ₁₅ CHN

Calculated: 65.26 6.22 2.98

F .: 65.16 6.28 3.13

(b) Compound 2-methanesulfonic acid salt of 2'-N, N-dimethylglycyl-taxol

2 '- (N, N-Dimethylglycyl) taxol (0.06g, 0.064 mmol) is dissolved in t-butanol (2 mL) and water (1.5 mL). The mixture is cooled to about 5 ° C and treated dropwise with methanesulfonic acid (3.1 ml, 2 mg / ml, 0.0645 mmol). The mixture is then stirred for 1 minute at 0 ° C to 5 ° C and filtered through a 20-micron filter (Millipore) in a cooled with a dry ice-isopropanol mixture flask. The solution is freeze-dried. Obtained 0,058g product (88%), mp 170-173 ⁰ C. C ₅₂ H ₆₂ N ₂ SO ₁₈ -2H ₂ O CHN Calcd .: 57.83 6.27 2.6

found: 57.49 6.06 2.73

Physical Properties

Molecular weight: 1,035 F .: 170-173 ⁰ C (dec.)

Solubility: 15 mg / ml (light cloudy)

2 mg / ml (clear)

Investigation of chemical stability

The compound is subjected to stability tests according to the following methods.

The stability of derivatives is examined at 25 ° C and 37 ° C at different pH values. The plasma analyzes are carried out at 37 ° C in rat and human plasma. Human plasma is obtained from the Watkins Hospital and rat plasma from the Animal Care Unit of the University of Kansas. The used derivative concentration is about 20 to 25 pg / ml. A stock solution of the compound is prepared in a concentration of 0.8 to 1.0 mg / ml and added to plasma to obtain the desired concentration (20-25MgZmI). ΙΟΟμΙ samples are taken and quenched with 250μΙ acetonitrile and centrifuged to precipitate the plasma proteins. The degradation kinetics are examined by HPLC plotting the peak area versus time. The t ₉₀ and tso values are calculated. Both the chemical studies and the

Plasma analyzes are performed by high performance liquid chromatography using a RP-8 column (15 cm) and a precolumn (5 cm). The detector is set to 227 nm. The mobile phase consists of 0.02M acetate (pH 5), acetonitrile 50:50 or 35:65, and the flow rate is in the range of 1 to 1.5 ml / min, or the same solvents are included to 0.001 M tetrabutylammonium hydrogen sulfate at a flow rate of 1 ml / min.

In the stability studies, the disappearance of the compound peak results in the formation of a peak having a retention time equal to that of taxol. The identity of this peak is further confirmed by degradation studies of the new derivatives. Thus, 2 '(DMG) taxol is incubated with water at 37 ° C and the product is evaporated and purified by preparative thin layer chromatography. After purification, the product is analyzed by HPLC and spectroscopic methods. The product shows a molecular ion peak at m / e 860 (M + Li) ⁺ , indicating that the product is taxol.

HPLC operating conditions RP-8,150 mm length, 4.6 mm inner diameter t _1/2 (hrs) Pillar: 0.02M acetate (pH 5): acetonitrile 50:50 mobile phase: Kratos Spectroflow757 96.2 Detector: 1 ml / min 55.4 Flow rate: 11.2 ml of compound 2 Retention time: 5.5 ml of taxol 89.8 Results of chemical stability studies conditions t ^ imine) 0.02M acetate (0.1mg / ml) 3.05 (pH 3,5,25 ° C) 121.6 (PH Wert4,5,25 ° C) 198.6 Water (2 mg / ml) (pH value 3,8,37 ⁰ C) Plasma stability, 37 ° C conditions Rat plasma (20 μ / ml) Dog plasma (20 μ / ml) Human plasma (20 u / ml)

Example Il

Salts of 2 '- (N, N-diethylaminopropionyl) taxol

CCH,

3.R = O = C-CH ₂ -CH ₂ N (C ₂ Hs) ₂ • HCI R '= H

4, R = O = C-CH ₂ -CH ₂ N (C ₂ H ₅ I ₂ .CH ₃ SO ₃ HR '= H

(a) Compound 3-HCl salt of 2 '- [3- {N, IM-diethylamino) -propionyl] -taxol

A solution of taxol (0.12 g, 0.14 mmol) in CH ₂ Cl ₂ (12 mL) containing N, N-diethylaminopropionic acid hydrochloride (0.025 g, 0.145 mmol) is added with IS-dicyclohexylcarbodiimide (0.08 g, 0.38 mmol ) and 4-dimethylaminopyridine (0.01 g, 0.081 mmol). The resulting mixture is stirred for 24 hours at room temperature. The reaction mixture is filtered and the filtrate is evaporated under nitrogen. The crude material is chromatographed on a silanized silica gel column (18 g, 22 cm) and eluted successively with ethyl acetate, petroleum ether 1: 2, ethyl acetate, petroleum ether 1: 1 and ethyl acetate. The ethyl acetate-petroleum ether fractions form a solid precipitate on slow evaporation, which is filtered. The product-containing mother liquors are combined, evaporated, treated to dryness with petroleum ether and allowed to stand. The product is filtered off. 0.068 g of product (48%) is obtained. F. 188-191 ⁰ C. Mass Spectrum (FAB) m / e 981 (M + H) ⁺ . The NMR spectrum (300Hz, CDCl ₃ ) shows that the resonance of the 2'-proton is 4.71 ppm for taxol

5.53ppm is shifted. The N-ethyl groups show the methyl resonance at 1.0ppm and the CH _r resonance at 2.52ppm. All other resonant properties of the expected compounds are observed. C ₅₄ H ₆₅ CIN ₂ O ₁₅ CHN

calculated: 63.73 6.43 2.75

Filled: 64.84 6.84 2.89%.

Physical properties (for compound 3) Molecular weight: 1017.56

F .: 186-189 ° C (Zers.)

Solubility: -0.8 mg / ml

HPLC conditions

Column: RP-8,150 mm length, 4.6 mm inner diameter

mobile phase: 0.02M acetate (pH 5): acetonitrile 35:65

Detector: KratosSpectroflow757

Flow rate: 1.5 ml / min

Retention volume: 16.71 ml (compound 3)

Chemical stability

Conditions: t _1/2 (hrs)

0.02M acetate

(0.01 mg / ml, pH3.5, 25 ° C) 438.6

0.02M phosphate

(0.02 mg / ml, pH 7.4.25 ° C) 0.25

Plasma stability, 37 ° C:

Conditions t _1/2 (min)

Human plasma 4.2

(b) Compound 4-methanesulfonic acid salt of 2 '- (N, N-diethylaminopropionyl) taxol To prepare the methanesulfonic acid salt of Ν, Ν-diethylaminopropionic acid, 10 g of QAE-Sephadex (Pharmacia) are swollen in 0.1 M NaCl for 75 hours. 75% of this material is poured into a column. The column is washed with distilled water (700 ml). The column is then equilibrated with 500 ml of CH ₃ SO ₃ Na (prepared from 0.5M methanesulfonic acid and 0.5M sodium hydroxide and titrated to pH 6). The complete disappearance of Cl "is tested by recovering the eluate and testing for Cl" by adding a few drops of 1% silver nitrate in a few drops of nitric acid. The column is further washed until neutral with distilled water.

2.5 g of Ν, Ν-Diethylaminopropionsäure-HCl salt in 15 ml of water are placed on the column and eluted with water. Four 50 ml fractions are recovered. The first few fractions contain the product. These fractions are combined and the solvent is removed. The residue is dissolved in a mixture of methylene chloride and ethanol and dried over magnesium sulfate. After removal of the solvent, 3.1 g of product. The product is precipitated from ethanol and ether. 2.6 g methanesulfonic acid salt of Ν, Ν-diethylaminopropionic acid are obtained.

To a solution of taxol (0.05 g, 0.058 mmol) and Ν, Ν-diethylaminopropionic acid methanesulfonic acid salt (0.014 g, 0.058 mmol) in methylene chloride (10 mL) is added.!, S-dicyclohexylcarbodiimide (0.061 g, 0.3 mmol). The mixture is stirred at room temperature for 24 hours. The reaction mixture is filtered and the filtrate is evaporated under nitrogen. The residue is chromatographed on a silanized silica gel column and eluted with ethyl acetate, petroleum ether 1: 1 and ethyl acetate. The ethyl acetate / petroleum ether fractions give, on slow evaporation, 0.048 g product (74%). F. 170-174 ⁰ C.

Physical Properties 1,077.12 Molecular weight: 170-174 ⁰ C F .: > 10mg / ml Solubility: > 99% HPLC purity: Chemical stability t _1/2 (hrs) conditions 0.02M acetate 305 (pH value 4,5,25 ° C) 0.02M acetate 20.7 (pH value 5,25 ° C)

Example III

Preparation of 7- (N, N-dimethylglycyl) taxol or a salt thereof

5.R = O = CO-CH ₂ -CCI ₃ R ' 6.R = O = CO-CH ₂ -CCI ₃ R ' 7.R = H R ' 8.R = H R '

= H

= O = C-CH ₂ -N (CH ₃ I ₂

= O = C-CH ₂ -N (CH ₃ J ₂

= O = C-CH ₂ -N (CH ₃ I ₂ CH ₃ SO ₃ H

(a) 2 '- (troc) -taxol (compound 5)

Taxol (0.27 g, 0.316 mmol) is dissolved in CH ₂ Cl ₂ (10 mL) and pyridine (1.5 mL). The reaction mixture is cooled to -20 ⁰ C to -25 ° C and treated with chloroformate ^^^ - trichlorethylester (80μΙ). The reaction mixture is stirred for 3 hours at the temperature mentioned. Another 25 ml of chloroformates are added and the reaction mixture is stirred overnight. The reaction mixture is then diluted with CH ₂ Cl ₂ (50 ml) and washed successively with 0.1 N HCl (2 × 25 ml) and 0.05 M cold NaHCO ₃ (1 × 25 ml) and water. The organic extract is dried over anhydrous MgSO 4 and the solvent is removed. The crude material is purified by preparative thin layer chromatography on silanized silica gel plates using ethyl acetate-petroleum ether 1: 3 as a run. A band above Taxol is excised and eluted with ethyl acetate. After removal of the solvent, 0.32 g (97%) of product mp. 221-226 ⁰ C (dec., Softening at 160 ⁰ C).

(b) 2 '- (TROC) -7- (N, N-dimethylglycyl) taxol (Compound 6)

A mixture of 2 '- (troc) -taxol (0.27 g, 0.262 mmol) and Ν, Ν-dimethylglycine (0.054 g, 0.524 mmol) is dissolved in CH ₂ Cl ₂ (15 ml). This solution is added with!, S-dicyclohexylcarbodiimide (0.215 g, 1.04 mmol) and 4-dimethylaminopyridine (0.025 g, 0.2 mmol). The mixture is stirred for 2 days at room temperature. The reaction mixture is filtered and the solvent removed. The crude material is purified by preparative thin layer chromatography on silanized silica gel plates using ethyl acetate: petroleum ether 1: 1 as eluent. A band below Taxol (R f 0.47, ethyl acetate: petroleum ether 1: 1) is excised and eluted with ethyl acetate. After removal of the solvent, 0.26 g of product (89%) of mp. 176 ° C to 180 ° C (dec.). C ₅₄ H ₆₀ Cl ₃ N ₂ O ₁₇ CHN

reported: 58.1, 5.42, 2.51

F .: 58.68 6.00 3.18%.

(c) 7- (N, N-dimethylglycyl) taxol (Compound 7)

To a solution of 2 '- (troc) -7- (N, N-dimethylglycyl) taxol (0.335g, 0.3mmol) in methanok acetic acid 9: 1 (12ml) is added zinc dust (0.275g). The mixture is stirred for 25 minutes at room temperature and then filtered. The filtrate is concentrated to -1 ml and diluted with CH ₂ Cl ₂ (35 ml) and washed successively with 0.01M HCl (2 × 20 ml), 0.01M cold NaHCO ₃ and water. The organic extract is dried over anhydrous Na ₂ SO ₄ . After removal of the solvent, 0.24 g of product are obtained. This compound is purified by preparative thin layer chromatography on silanized silica gel plates (3 x 20 cm x 20 cm) using CH ₂ Cl ₂ : ethyl acetate 7: 1 as eluent. The band corresponding to 7- (DMG) taxol (Rf 0.35) is excised and eluted with ethyl acetate and ethanol. After removal of the solvent to obtain 0.19 g of product (68%) of F. 180 ⁰ C to 185 ° C (dec., Softening at about 140 ° C). Mass Spectrum (FAB): m / e 939 (M + H) ⁺ . In the NMR spectrum (300 MHz, CDCl ₃₎ , the resonances of 7-H at 4.33 ppm in taxol appear as a doublet doublet at 5.65 ppm The N- (CH ₃ ) ₂ resonance occurs as a singlet at 2.35 The methylene group of glycinate is found at 3.16 ppm.

(d) Methanesulfonic acid salt of 7- (dimethylglycyl) taxol (Compound 8)

Dissolve 7- (dimethyl glycyl) taxol (0.065 g, 0.069 mmol) in t-butanol (2.5 mL) and water (1 mL). The solution is cooled to 5 ° C to 10 ° C and treated with methanesulfonic acid (3.36ml, 2mg / ml, 0.0697 mmol). The mixture is stirred for 2 minutes and

Filtered through Millipore filter in an ice-cooled flask. The filtrate is freeze-dried. This gives 0.066 g of product (94%) of mp. 164 ⁰ C to 168 ° C (decomp.)

C ₅₂ H ₆₂ N ₂ O ₁₈ S-2H ₂ O: CHN calc .: 58.29 6.19 2.6

F .: 58.05 6.00 1.72.

Physical Properties

Molecular weight:

Solubility:

HPLC conditions

Pillar:

mobile phase:

Detector:

Flow rate:

Retention volume:

Chemical stability

conditions

0.02M acetate (pH 3.5.25 ° C) 0.02M acetate (pH 4.5.25 ° C) 0.02M phosphate (pH 7.4.25 ° C )

Plasma stability, 37 ^° C

Conditions Rat plasma (20 μg / ml) Human plasma (20 μg / ml) Human plasma (10 μg / ml)

Example IV Preparation of 2 ', 7- (dimethylglycyl) taxol

1035

164 ° C to 168 ° C> 2 mg / ml

RP-8.150 mm length, 4.6 mm inner diameter 0.02 m acetate (pH 5): acetonitrile 35:65 Kratos-Spectrof low 757 1.5 ml / min 15.07 ml (compound 8)

t _1/2 (hrs.) 3397 1719 33.8 hrs.

ti / ₂ (hrs) 17,3 27,7 24,4

9. R = R '= O = C-CH ₂ -N (CHa) ₂

Taxol (0.06 g, 0.0702 mmol) is dissolved in anhydrous methylene chloride (5 mL) and N, N-dimethylglycine (0.015 g, 0.145 mmol) is added. To this mixture is added IS-dicyclohexylcarbodiimide (0.08 g, 0.388 mmol) and 4-dimethylaminopyridine (0.008 g, 0.065 mmol). The mixture is stirred at room temperature for 24 hours and filtered. From the filtrate, the solvent is removed. The residue is purified by preparative thin layer chromatography on silanized silica gel plates using ethyl acetate: 1: 1 ethyl acetate as eluent. A band (Rf value 0.17) above dimethylaminopyridine is scraped off and eluted with a mixture of ethyl acetate and ethanol. The solvent is removed. The residue is recrystallized from ethyl acetate-petroleum ether. Obtained 0,046g product (64%), melting at 19 ° C to 198 ⁰ C. Mass spectrum: m / E1024; (M ⁺ ). In the NMR spectrum (300MHz, CDCI ₃ ), the C ₂ proton (4.71 ppm) and the C? Proton (4.33 ppM) shift to 5.5 ppm and 5.6 ppm, respectively, resulting in esterification in the 2'- and 7-position indicates. Further, the N-CHs proton as a singlet occurs at 2.3 ppm

C ₅₅ H ₆₆ N ₃ O ₁₆ : CHN

calculated: 63.39 6.48 4.03

Filled: 63.00 6.98 3.98%.

Preparation of dimethanesulfonic acid salt of 2 ', 7- (N, N-dimethylglycyl) taxol A solution of 2', 7- (dimethylglycyl) taxol (60 mg, 0.0585 mmol) of intert. Butanol (2 ml) and water ( 1 ml) which is cooled to 0 ⁰ C to 5 ⁰ C is added methanesulfonic acid (11.37 mg, 3.79 ml, 0,117mMol). The mixture is stirred at ⁰ ° C. to 5 ° C. for 2 minutes and then filtered through a Millipore filter (0.2 μm). The filtrate is freeze-dried. This gives 62 mg of product, mp. 160 ° C to 163 ° C.

Physical Properties

Molecular weight:

Solubility:

HPLC purity:

HPLC operating conditions

Pillar:

mobile phase:

Detector:

Flow rate:

Retention volume:

1217

160 ° C to 163 ° C (dec.)> 10 mg / ml -96%

Rp-8,150 mm, 4.6 mm inside diameter

0.02M acetate (pH 5): acetonitrile 50:50 containing 0.005m TBA

Kratos Spectroflow 757

1 ml / min

6.64 ml

5,8ml (Taxol)

Example V Preparation of 7- (L-alanyl) taxol or a salt thereof

(A) 2 ', 7-di (tert-Boc.-L-alanyl) -taxol

A solution of taxol (0.21 g, 0.246 mmol) and N-tert-Boc-L-alanine (0.14 g, 0.739 mmol) in methylene chloride (15 mL) is added with!, S-dicyclohexylcarbodiimide (0.25 g, 1 , 21 mmol) and dimethylmonopyridine (0.025 g, 0.2OmMoI). The mixture is stirred at room temperature for 24 hours and filtered. The residue is chromatographed on a silanized silica gel column (20 g, 14 cm) and eluted with ethyl acetate, petroleum ether 1: 1 and ethyl acetate. The ethyl acetate-petroleum ether fractions containing the disubstituted derivative are combined. The solvent is removed. This gives 0.27 g of compound (92%) of mp 158-161 ° C (dec.).

(b) 7- (L-alanyl) taxol

2,7 Di- (tert-Boc.-L-alanyl) taxol (0.29 g, 0.242 mmol) and formic acid (2 ml) are mixed and stirred at room temperature for 40 minutes. Excess formic acid is removed under nitrogen. The residue is dissolved in ethanol and treated with petroleum ether. The solid is filtered off. This gives 0.27 g of 2 ', 7-di (alanyl) taxol.

The crude dialanyl derivative thus obtained is taken up in acetonitrile (4 ml) and phosphate buffer (0.02 m, pH 7.4, 50 ml), and the mixture is stirred for 12 hours at room temperature. The pH of the solution is raised to 6.8 with a few ml of 5% Na ₂ HPO ₄ . The cloudy solution is stirred for a further 8 hours at room temperature. The reaction mixture is then diluted with methylene chloride (50 ml) and treated with cold NaHCO ₃ (0.05 m, 50 ml). The reaction mixture is extracted immediately with methylene chloride (3x 50 ml), and the organic extract is washed once with water and dried over anhydrous sodium sulfate. After removal of the solvent gives 0.24 g of product. This compound is purified by column chromatography on a silanized silica gel acid. This gives 0.135 g of product (63%). (Purity> 95%). F. 159 ° C to 163 ° C. Mass Spectrum (FAB): m / e 925 (M + H) ⁺ . In the NMR spectrum (300 MHz, CDCl ₃ ), the 7 H at 4.33 ppm in taxol appears as a doublet doublet at 5.65 ppm. The CH ₃ group on the alanine residue appears as a doublet at 1.27 ppm. C ₆₀ H ₆₈ N ₂ O ₁₆ -2.5H ₂ O: CHN

calculated: 61.91, 6.54, 2.89

F .: 61.41 6.59 2.78%.

(c) Methanesulfonic acid salt of 7- (alanyl) taxol

A solution of 7- (alanyl) -taxol (62 mg, 0,0658mMol) in tert-butanol (2 ml) and water (1 ml) was cooled to 0 ⁰ C to 5 ⁰ C and with methanesulfonic acid (6.39 mg, 2.13 ml, 3 mg / ml). The mixture is stirred for 2 minutes at this temperature and then filtered through a Millipore filter (0.2 μm). The filtrate is freeze-dried. This gives 66 mg of product of F. 180 ° C to 184 ° C.

Physical Properties

Molecular weight:

Solubility:

HPLC operating conditions

Column: mobile phase:

Detector:

Flow rate:

retention volume

1021

180 ° C to 184 ° C (dec.)> 2 mg / ml

RP-8,150 mm length, 4.6 mm inner diameter

0.02M acetate (pH 5): acetonitrile 50:50 containing 0.001M

tetrabutylammonium hydrogen

Kratos Spectroflow 757

1 ml / min

8.7 ml

7.3 (Taxol)

Plasma stability, 37 ° C

Conditions ti _{/ 2} (hrs)

Human plasma (20 μg / ml) 11.9

Example Vl Preparation of 2 '- (alanyl) taxol

(a) Preparation of 2 '- (CBZ-L-alanyl) taxol

To a solution of taxol (30 mg, 0.0036 mmol) and CBZ-L-alanine (8.5 mg, 0.036 mmol) in methylene chloride (5 ml) is added DCC (45 mg) and 4-dimethylaminopyridine (4 mg). The mixture is stirred for 3 days at room temperature. The reaction mixture is filtered and the solvent is removed from the filtrate. The residue is developed by preparative thin-layer chromatography on silanized silica gel plates using ethyl acetate-petroleum ether 1: 1 as eluent. The band above Taxol is excised and eluted with ethyl acetate. Removal of the solvent gives 28 mg of 2 '- (CBZ-L-alanyl) taxol.

(b) Preparation of 2 '- (alanyl) taxol by deprotection of 2' - (CBZ-L-alany1) taxol

2 '- (CBZ-L-alanyl) taxol is dissolved in ethanol in the presence of an organic acid such as acetic acid or formic acid and stirred for 2 hours at room temperature in the presence of 5% palladium on carbon. The reaction mixture is filtered to remove the catalyst and the solvent is removed. The crude product is dissolved in ethanol and treated with petroleum ether. 2'- (Alanyl) taxol is obtained as formate or acetate salt in low to moderate yield.

Example VII Preparation of 2 '- (lysyl) taxol

(a) Preparation of 2 '- (N-di-tert-Boc-lysyl) -taxol

A mixture of taxol (30 mg, 0.035 mmol) and N-di-tert-Boc-L-lysine (19 mg, 0.0368 mmol) in methylene chloride (10 ml) is treated with DCC (100 mg) and 4-dimethylaminopyridine (10 mg). added. The mixture is stirred for 2 days at room temperature and then filtered. Subsequently, the solvent is removed from the solution. The residue is purified by preparative thin-layer chromatography on silanized silica gel plates using ethyl acetate-petroleum ether 1: 1 as eluent. The band above Taxol is excised and eluted with ethyl acetate. After removal of the solvent, 20 mg of product.

(b) Preparation of 2 '- (lysyl) taxol by removal of the tert-Boc group

The N-tert-Boc-protected amino acid derivative of Taxol is reacted with formic acid (99%, Sigma) for 30 to 40 minutes at room temperature. The excess formic acid is removed by evaporation under nitrogen. The crude material is purified by crystallization or chromatography to give the N-unprotected amino acid derivative of taxol as a formate salt.

Example 8 Preparation of 2 '- (L-alanyl) taxol

(a) Preparation of 2 '- (FMOC-L-alanyl) taxol

To a solution of taxol (60 mg, 0.072 mmol) and N-FMOC-alanine (22.4 mg) in methylene chloride (6 ml) is added DCC (60 mg) and 4-dimethylaminopyridine (2 mg). The mixture is stirred for 2 days at room temperature and then filtered. The solvent is removed from the filtrate. The crude material is purified by preparative thin layer chromatography on silanized silica gel plates using ethyl acetate-petroleum ether 1: 2 as eluent. The band above Taxol is excised and eluted with methylene chloride. After removal of the solvent to obtain 48mg 2 '- (FMOC-L-alanyl) -taxol from mp 162 ° C to 164 ° C (dec.).

(b) Removal of the FMOC group from the protected amino acid derivative of Taxol

The N-FMOC protected amino acid derivative of taxol is reacted with piperidine in methylene chloride for 2 hours. Subsequently, the solvent is removed. The residue is purified by chromatography. The deprotected amino acid derivative of taxol is obtained.

Pharmaceutical preparations

The compounds of the invention can be used as such in pharmaceutical preparations or in the form of pharmaceutically acceptable salts, in particular in the form of non-toxic pharmaceutically acceptable acid addition salts or compatible basic salts. These salts can be prepared from the compounds of the invention according to conventional chemical methods. Normally, the salts are prepared by reacting the free base or acid with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or combinations of different solvents. For example, the free base may be dissolved in an aqueous solution of the appropriate acid, and the salt recovered by conventional means, for example by evaporation of the solution. Another possibility is to use the free base in an organic solvent, e.g. A lower alkanol, an ether, an alkyl ester or mixtures thereof, such as methanol, ethanol, ether, ethyl acetate, ethyl acetate-ether solution and the like, after which the solution is treated with the appropriate acid to form the corresponding salt , The salt is recovered by conventional recovery methods, for example, by filtration of the desired salt after spontaneous separation from the solution, or it can be precipitated and recovered by adding a solvent in which the salt is insoluble.

The taxol derivatives of the invention can be used for the treatment of cancers because of their cytotoxic antitumor activity. The novel compounds may be administered in the form of tablets, pills, powder mixtures, capsules, injectables, solutions, suppositories, emulsions, dispersions, feed premixes, and in other suitable forms. The pharmaceutical preparations containing this compound are conveniently mixed with a non-toxic, pharmaceutical, organic carrier or a non-toxic, pharmaceutical, inorganic carrier, usually in an amount of from about 0.01 mg to 2500 mg or more per Dose unit, in particular in an amount of 50 to 500mg. Typical examples of pharmaceutically acceptable carriers are mannitol, urea, dextranes, lactose, potato and corn starches, magnesium stearate, talc, vegetable oils, polyalkylene glycols, ethylcellulose, poly (vinylpyrrolidone), calcium carbonate, ethyl oleate, isopropyl myristate, benzyl benzoate, sodium carbonate, gelatin, potassium carbonate , Silica and other conventionally used compatible carriers. The pharmaceutical preparation may also contain non-toxic auxiliary substances, e.g. Emulsifiers, preservatives, wetting agents and the like, such as sorbitan monolaurate, triethanolamine oleate, polyoxyethylene monostearate, glyceryl tripalmitate, dioctyl sodium sulfosuccinate and the like.

An example of a typical method for preparing a tablet containing such an agent is to first mix the active ingredient with a non-toxic binder such as gelatin, mucilage gum arabic, ethyl cellulose or the like. The mixing is conveniently carried out in a conventional V-mixer and generally under anhydrous conditions. Subsequently, the mixture produced in this way can be granulated with conventional tabletting machines and processed into tablets. The freshly prepared tablets can be provided with a coating or can remain uncoated. Examples of suitable coatings are non-toxic coatings such as shellac, methylcellulose, carnauba wax, styrene-maleic acid copolymers and the like. For oral administration, compressed tablets containing 0.01 mg, 5 mg, 25 mg, 50 mg, 500 mg and the like up to 2,500 mg of active ingredient-active ingredients as described above and according to known production techniques (see Remington's Pharmaceutical Science, Chapter 39, Mack Publishing Co., 1965).

To prepare the tablets, for example, the active ingredient, corn starch, lactose, dicalcium phosphate and calcium carbonate are uniformly mixed under dry conditions in a conventional V-mixer. Subsequently, a 10% corn starch paste is prepared and uniformly mixed with the mixture obtained above. The mixture is then passed through a conventional light mesh screen, dried in an anhydrous atmosphere and then mixed with calcium stearate, compressed into tablets and optionally coated. Further tablets with an active ingredient content of 10, 50, 100 and 150 mg are prepared in a similar manner. The following Preparation I is an example of a tablet preparation containing a compound of the invention.

Preparation I per tablet, mg ingredients 50.0 active substance 15.0 corn starch 4.5 Corn starch paste 15.0 calcium carbonate 67.0 lactose 2..O calcium stearate 50.0 dicalcium

The preparation of capsules having an active ingredient content of 10 to 2500 mg for oral use consists essentially in mixing the active ingredient with a non-toxic excipient and enclosing the mixture in a polymeric shell, generally gelatin or the like. The capsules may be known soft capsules prepared by entrapping the compound in the form of an intimate dispersion with an edible, compatible carrier. Or they may be hard capsules which essentially contain a compound of the invention 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 a compound of the invention, alone or in admixture of two or more of these compounds, are prepared, for example, in the following manner:

Preparation Il per capsule, mg ingredients 50.0 active substance 100.0 calcium carbonate 200.0 Lactose, U.S.P. 130.0 Strength 4.5 magnesium stearate

The above ingredients are mixed in a conventional mixer and then filled into commercial capsules. When using higher concentrations of active ingredient, the amount of lactose is reduced accordingly. The compounds of the invention may also be subjected to lyophilization and optionally processed with other pharmaceutically acceptable carriers into formulations suitable for administration by parenteral injection. For such administration, the preparation is reconstituted in water (usually saline) or in a mixture of water and an organic solvent such as propylene glycol, ethanol and the like. The dose administered (single dose, multiple dose or daily dose) will of course vary depending on the specific compound of the invention employed, due to the differential potency of this compound, the route of administration chosen, the body weight of the recipient and the condition of the patient. The dose administered

is not subject to any specific restrictions. In general, however, it is an effective dose or one molar equivalent of the pharmacologically active free form resulting from a dosage preparation in the metabolic release of the active ingredient to achieve the desired pharmacological and physiological effects. The dosage administered is generally in the range of 0.8 to 8 mg / kg body weight or within the range of about 50 to 275mg / m ² of the patient, in particular in the range of about 230 to 275 mg / m ^2.

Biological activity

As mentioned above, the taxol derivatives of the invention possess valuable anti-tumor activity. The compounds are particularly useful in the treatment of the same cancers for which taxol has been shown to be effective, including human lung tumors, melanoma, leukemia, breast tumors and colon cancer.

The biological activity of the taxol derivatives has been studied by (A) in vitro studies to measure microtubule assembly kinetics; (B) in vitro studies of the kinetics of B 16 melanoma cell cultures; and (C) In vivo studies on SRC subrenal capsule xenograft (SRC) from MX-1.

(A) In vitro study of microtubule assembly kinetics

Mikroltubili represent a component of eukaryotic cells. The structure of the microtubules is closely related to cell division and cell proliferation. It has been shown that microtubule polymerization is very sensitive to calcium; Calcium can inhibit tubulin synthesis and depolymerize already established microtubules. Known antitumor compounds were tested for their effect on microtubule building. Vinca alkaloids, such as vinblastine and vincristine, have a destructive effect on cellular microtubules, i. H. it has been demonstrated in vitro to inhibit microtubule assembly and depolymerize existing microtubules.

Similarly, colchicine has been shown to depolymerize microtubules in cells. On the other hand, taxol has been shown to have a very specific mechanism of action. Taxol promotes microtubule assembly, but inhibits dissection, interacting with the G ₂ and M phases of cell cycles and cell division. In vitro studies have shown that microtubules already polymerized in the presence of taxol resist depolymerization by other drugs, such as CaCl ₂ , or by cold temperatures, which typically induce microtubule depolymerization.

According to the invention, studies were carried out to investigate the effect of taxol derivatives on the construction of microtubules. Examination of the microtubule structure was performed using the 2 'and 7 derivatives according to the described in vitro methods; see. For example, Mellado et al. Biochemical and Biophysical Research Communications, Vol. 124, No. 2 (1984), pages 329-336; Margri et al., J. Org. Chem., Vol. 51 (1986), pages 797-802; and Parness et al. Biochemical and Biophysical Research Communications, Vol. 105, No. 3 (1982) pp. 1082-1091. The ability of these compounds to build microtubules is as follows: taxol> 7- (N, N-dimethylglycyl) taxol (11)> 2 '- (N, N-diethylaminopropionyl) taxol (8)> 2'- (N, N-dimethylglycyl) taxol (2). This study shows that the free 2'-hydroxyl group is essential for the construction of microtubules. The 2'-derivatives are only active when the 2'-hydroxyl group is released in the course of the study. The 7-derivatives have a free 2'-hydroxyl group and are therefore active. This result agrees well with the activities of 2'- and 7-acetyltaxol reported in the literature (see Melado et al., Biochemical and Biophysicyl Research Communications, Vol. 124, No. 2 [1984], pages 324-336).

(B) In vitro study on B-16 cell cultures

In order to confirm the activity of the taxol derivatives of the invention, in vitro studies were performed on cell cultures using B-16 melanoma cells. These investigations were carried out according to customary methods, cf. Donoso and Hirnes, Cancer Biochem. Biophys., Vol. (1984), page 133.

In investigating the use of B-16 melanoma cells, the order of potency was as follows: Taxol> 2 '- (N, N-dimethylglycyl) -taxol> 2' - (N, N-diethyl-monopropionyl) - taxol> 7- ( N, N-Dimethylgylcyl) -taxol. This study and other kinetic studies indicate that the 2'-derivatives are converted to taxol and display activity. Therefore, the 2'-derivatives probably act as drug precursors. On the other hand, the 7-derivatives have their own activity and apparently do not act as drug precursors.

(C) in vivo studies

The third type of study was performed to confirm the biological activity of taxol derivatives in an in vivo study in mice with a foreign implant of subrenal capsular human mammary carcinoma MX-1. The procedure was as reported by NIH Publication No. 84-2635, In Vivo Cancer Model (February 1984) pages 23-24.

method

In this method, test groups of mice (6 mice per test group) and control groups of 12 mice per group were used. A tumor fragment (human breast carcinoma MX-1) was implanted into the individual mice under the membranous cover of the kidney.

The following test schedule was adhered to:

Day 0: stunning the animals; Recording the body weight (weight day 1); Implanting the tumor, measuring and recording; Randomize the animals after recovery from anesthesia; Preparation of bacterial cultures; Determination of the solubility of the test substance; Register the daily deaths.

Day 1: Check the cultures; Discarding if they are contaminated; Preparing the test materials; Beginning with the injections of the test substances (in the neck) on the basis of the respective body weight; the treatment takes place on days 1, 5 and 9 (QUD); fresh production of the test substance on each day of injection and administration on the basis of the individual body weight on the respective day.

Day 2: reconsideration of cultures; Stop the test if the crops are contaminated and report accordingly.

Days 5 and 9: Preparation of fresh test substance on the respective day of injection and administration taking into account the individual body weight on that day.

Day 11: End and evaluation of the investigation; Record the body weight (weight day 2). Tumor measurement in OMU (ocular micrometer unit) - 100MU = 1 mm) and record.

evaluation

The measured parameter is the average change (delta) in body weight in terms of length and width measurement in mm. The average body weights of the animals are calculated on day 1 and day 11. The T / C values for all test groups with> 65% survivors on day 11 are calculated. An excessive change in body weight (test minus control) can also be used to assess toxicity. The dimensions are measured and recorded in eyepiece micrometer units (OMU). The following operations use a computer:

1) Conversion of OMU in millimeters (mm).

2) Calculation of the tumor weight (mg) from the tumor dimensions (mm χ mm) according to the following formula for the volume of a stretched ellipsoid:

Lxw ²

Where L represents the longer of the two dimensions.

3) Calculation of the change (delta) of the average tumor weight for each group of mice: change in the average tumor weight = (average tumor weight _ENDE- average tumor weight beginning) ·

4) Calculation of the change (delta) of the average tumor weight for the test groups (T) and the control groups (C).

5) Calculate the T / C% values for all test groups with> 65% survivors on the last valuation day:

T / C%

AWtT AWtC

• x 100-ifAWtT is positive;

AWtT

T / C% = - x 100-ifAWtT is negative

average tumor weight EGN

Criteria for the activity

An initial T / C value of 20% is considered necessary for demonstrating moderate activity. A reproducible T / C value of S 10% is considered to be a significant activity.

Results

The results of the tests carried out with various taxol derivatives are shown in Tables I and II.

Table I Human breast carcinoma tests

treatment connection dose % T / C Results of average tumor weights Default- The End default weight number of Testkör Tierkör- NSC (Mg / kg) control deviation deviation Variegated to survive perge perge- control 0.00 TOX ^b beginning 0.1 mg 2.4 tion the animals / wiht- wichts- group taxol 22.40 CR ^C 0.1 12.79 0.0 (delta) total number modification difference No. Ta xo I 15.00 -84 mg 0.1 0.00 0.0 12.04 11/12 0001 taxol 10.00 CR 0.75 0.1 0.00 0.1 3.6 -2.6 -0.9 0102 taxol 6.67 CR 0.60 0.1 0.09 0.0 -0.81 6.6 -4.2 -2.5 0103 taxol 4.45 TOX 0.81 0.1 0.00 0.0 -0.49 6.6 -2.0 -0.3 0104 Verb 1 " 30.00 CR 0.58 0.1 0.00 -0.74 5.5 -1.5 0.2 0105 Verb 1 ^a 20.00 CR 0.74 0.0 Ό, ΟΟ 0.0 -0.79 5.5 -1.2 0.5 0106 Verb 1 ^a 13,30 -53 0.79 0.1 0.00 0.0 1.6 -3.8 -2.1 0207 Verb 1 " 8.90 -78 0.68 0.1 0.00 0.4 -0.56 4.6 -2.2 -0.5 0208 Verb.T 5.90 0.56 0.1 0.36 0.1 -0.64 4.6 -2.3 -0.6 0209 0.64 0.13 -0.41 5.6 -0.9 0.08 0210 0.77 -0.48 6.6 -2.2 -0.5 0211 0.61

Compound 1 -2 '- (N, N-dimethylglycyl) -taxol ^b TOX means a toxic concentration ^c CR means a complete decrease of the tumor

Table II Human Mammary Carcinoma Tests

treatment connection dose % T / C Results of average tumor weights Default- The End default weight number of Testkör- Tierkör- NSC (Mg / kg) TOX ^C deviation deviation Variegated to survive perge- perge- taxol 15.00 TOX beginning 0.1 mg tion the animals / wichts- wichts- group taxol 7.50 CR ^d 0.1 0.0 (delta) total number modification difference No. taxol 3.75 5 mg 0.1 0.00 0.0 0/5 0310 taxol 1.88 CNTL 0.80 0.1 0.00 1.5 3.5 -1.7 2.3 0311 control 0.00 CR 0.82 0.1 1.51 2.2 -0.71 4.5 -2.9 1.1 0312 Verb 7 ° 33,00 -93 0.71 0.1 15.38 0.0 0.71 4.5 -1.0 3.0 0313 Verb.7 ^a 16.50 23 0.80 0.1 0.00 0.0 14.61 10/11 0001 Verb 7 " 8.25 34 0.77 0.1 0.04 1.5 -0.74 4.5 -2.7 1.3 0102 Verb 7 · 4.13 TOX 0.74 0.1 4.19 1.3 -0.55 3.4 -5.7 -1.7 0103 Verb 3 " 36,00 CR 0.59 0.1 5.66 3.38 4.4 -3.9 0.1 0104 Verb.3 ^b 18,00 CR 0.81 0.1 0.0 4.92 4.5 -4.5 -0.5 0105 Verb.3 ^b 9.00 CR 0.74 0.1 0.00 0.0 0/5 0206 Verb.3 ^b 4.50 0.75 0.1 0.00 0.0 -0.82 3.4 -3.3 0.7 0207 0.82 0.00 -0.87 5.5 -2.7 1.3 0208 0.87 -0.78 5.5 -2.8 1.2 0209 0.78

'Compound 7 = 7- (N, N-dimethylglycyl) -taxol

^b Compound 3 = HCl salt of 2 '- (3- (N, N-diethylamino) -propionyl) -taxol

^c TOX means a toxic concentration

^d CR means a complete decrease of the tumor

From the results, it can be seen that the taxol derivatives of the present invention have an excellent antitumor activity. These compounds are therefore suitable as antitumor agents because of their biological activity and their increased water solubility compared to taxol.

Claims (12)

in the R and R 'are each H or a group of the formula
C = O (Nl) R- wherein η is an integer of 1 to 3 and R ² and R ^{3 are} each hydrogen or an alkyl group of 1 to 3 carbon atoms, or wherein R ² and R ^{3 are taken} together with the nitrogen atom to which they are attached , form a saturated heterocyclic ring having 4 or 5 carbon atoms, with the proviso that at least one of R and R ^{1 is} not hydrogen, characterized in that taxol is reacted with a compound of formula IV
OH C = O (<? ^H 2> n (IV) R- in the n, R ² and R ^{3 have} the meaning defined above, and if appropriate reacting the resulting compound in a conventional manner to the corresponding salts. 2. The method according to claim 1, characterized in that one converts taxol and the compound of formula IV in an equivalent ratio of 1: 1, thereby obtaining a 2'-substituted compound of formula II in which R is a radical of formula III and R 'represents a hydrogen atom. 3. The method according to claim 1, characterized in that reacting 2 equivalents of the compound of formula IV with 1 equivalent of taxol, whereby one obtains a 2 ', 7-disubstituted compound of formula II in which the radicals R and R' in each case one Rest of the formula III mean. 4. The method according to claim 3, characterized in that subjecting the 2 ', 7-disubstituted compound of a cleavage reaction in which the 2'-substituent is cleaved, thereby obtaining a 7-substituted compound of formula II in which R is the Meaning H and R 'has a radical of the formula III. 5. The method according to claim 1, characterized in that one carries out the reaction in the presence of a condensing agent and in the presence or absence of a catalyst. 6. The method according to claim 4, characterized in that exposing the 2 ', 7-disubstituted compound to a pH of 7 to 7.4 in the cleavage reaction. 7. A process for the preparation of taxol derivatives of the formula II and the salts thereof CH ₃ CCO, O OR in which R and R 'each represent hydrogen atoms or the radical of an amino acid, with the proviso that at least one of the radicals R and R' does not represent a hydrogen atom, characterized in that one converts taxol with an N-protected amino acid and then the protective group of the amino acid removed. 8. The method according to claim 7, characterized in that one converts taxol and the N-protected amino acid in an equivalent ratio of 1: 1, thereby obtaining a 2'-substituted taxol derivative. A process according to claim 7, characterized in that taxol and the N-protected amino acid are reacted in an equivalent ratio of 1: 2 to give a 2 ', 7-disubstituted taxol derivative. 10. The method according to claim 9, characterized in that subjecting a compound of formula II to a cleavage reaction, whereby the 2'-Aminosäuresubstituent is cleaved off and receives a 7-amino acid taxol derivative. 11. The method according to claim 7, characterized in that the protective groups are removed by mild acid treatment. 12. A process for the preparation of taxol derivatives of the formula II and the salts thereof CH-, OR · O OH in which R 'is the radical of an amino acid from the group alanine, leucine, isoleucine, valine, phenylalanine, proline, lysine and arginine or a radical of the formula III C = O (III) in which η is an integer with a value of 1 to 3 and R ² and R ^{3 are} each hydrogen or an alkyl radical having 1 to 3 carbon atoms or in which R ² and R ³ together with the nitrogen atom to which they are attached, form a saturated heterocyclic ring having 4 or 5 carbon atoms, characterized in that (a) reacting taxol with a hydroxyl protecting group compound to form 2'-protected taxol; (b) reacting the 2'-protected taxol with alanine, leucine, isoleucine, valine, phenylalanine, proline, lysine, arginine or a compound of the following formula