WO2006067411A2

WO2006067411A2 - Process for preparing a phosphorylated colchinol derivative

Info

Publication number: WO2006067411A2
Application number: PCT/GB2005/004933
Authority: WO
Inventors: Simon Daniel Broady; Michael David Golden; John Leonard; James Campbell Muir; Aulelie Billard; Katie Murray
Original assignee: Angiogene Pharmaceuticals Ltd
Current assignee: Angiogene Pharmaceuticals Ltd
Priority date: 2004-12-23
Filing date: 2005-12-19
Publication date: 2006-06-29
Anticipated expiration: 2007-06-23
Also published as: WO2006067411A3; GB0428103D0

Abstract

Process for preparing a phosphorylated colchinol derivative of formula (I): wherein each R, which may be the same or different, is selected from (1-6C)alkyl, benzyl and C(O)(1-6C)alkyl, or two RO groups together form a (1-4C)alkylenedioxy group; by hydrolysing a compound of formula (II) wherein X is halogeno and each R is as hereinbefore defined. Processes for crystallising compounds of formula (I) and for preparing compounds of formula (II) are also described.

Description

CHEMICAL PROCESS

This invention relates to a chemical process for making phosphorylated colchinol derivatives which are vascular damaging agents, particularly N-acetylcolchinol-O-phosphate (ZD6126), and to intermediates used in the preparation of such compounds. The invention also relates to methods for obtaining pure ZD6126 in crystalline form.

Colchinol derivatives such as N-acetylcolchinol are useful in the treatment of diseases involving angiogenesis.

N-acetylcolchinol

(Ilia)

The use of colchinol and derivatives thereof as vascular damaging agents are described in International Patent Application publication number WO 99/02166.

A particular colchinol derivative which is described in International Patent Application publication number WO 99/02166 (Example 1 therein) is (5S)-5-(acetylamino)- 9,10,1 l-trimethoxy-όJ-dihydro-SH-dibenzofajCjcyclohepten-S-yl dihydrogen phosphate, also known as N-acetylcolchinol-O-phosphate and ZD6126, and which is referred to herein as ZD6126:

ZD6126

(Ia)

ZD6126 is a potent vascular targeting agent.

Generally it is not possible to convert a phenol such as N-acetylcolchinol directly into its phosphate derivative in high yield. A known method for preparing a phosphate derivative of phenols is to first convert the phenol group into a dibutyl phosphate ester and then convert this into the phosphate. This method is described for the preparation of ZD6126 in Patent Application publication number WO 99/02166. A considerable drawback with this 2-step procedure is that it involves the use of tetrazole, which is an explosive compound and not suitable for use in large scale manufacture.

The synthesis of ZD6126 by phosphorylation of N-acetylcolchinol with di-tert- butyldiethylphosphoramidite is described in Cancer Research, 62, 7247-7253 (2002).

WO 00/40529 suggests the phosphorylation of certain colchinol derivatives using phosphorus oxychloride in a chlorinated solvent such as trichloromethane. US 2004/ 138182 describes the preparation of colchine derivatives by the phosphorylation of colchinol with a phosphate ester in the presence of a non-aromatic amine.

There remains a continuing need for a safe, environmentally benign method for the preparation of the phosphorylated colchinol derivatives such as ZD6126 starting from N- acetylcolchinol. We have found a means for directly phosphorylating colchinol derivatives of the formula (II) defined hereinafter in high yield.

According to a first aspect of the present invention there is provided a process for the preparation of a phosphorylated colchinol derivative of the formula (I), or a pharmaceutically acceptable salt thereof:

(I) wherein each R, which may be the same or different, is selected from (l-6C)alkyl, benzyl and -C(O)(I -6C) alkyl, or two RO groups together form a (l-4C)alkylenedioxy group and Ac is acetyl; which process comprises hydrolysing a compound of formula (II)

(II) wherein X is halogeno (for example chloro) and each R is as hereinbefore defined. The hydrolysis may be carried out by hydrolysing the compound of formula (II) in an aqueous medium. Suitably the hydrolysis at a temperature of less than 35⁰C, for example from 20 to 3O⁰C.

Conveniently, the hydrolysis is carried out by quenching the reaction mixture with an aqueous medium, for example by adding the reaction mixture into the aqueous medium (e.g. water), or vice versa, to thereby effect hydrolysis of the compound of formula (II) directly. There follow particular and suitable values for certain substituents and groups referred to in this specification. These values may be used where appropriate with any of the definitions and embodiments disclosed hereinbefore, or hereinafter. For the avoidance of doubt each stated species represents a particular and independent aspect of this invention.

Suitable values for (l-4C)alkyl include methyl, ethyl, isopropyl, propyl, butyl, isobutyl and tertiarybutyl; suitable values for (l-6C)alkyl include (l-4C)alkyl, pentyl, cyclopentyl, hexyl and cyclohexyl; suitable values for -CO₂(I -4C)alkyl include -CO₂CH₃, -CO₂CH₂CH₃ and-CO₂tBu; suitable values for -CO₂(I -6C)alkyl include -CO₂(I -4C)alkyl and -CO₂pentyl, suitable values for -C(O)(l-4C)alkyl include -C(O)CH₃, and -C(O)CH₂CH₃; suitable values for -C(O)(I -6C)alkyl include -C(0)(l-4C)alkyl and -C0₂pentyl. When two RO groups together form a (l-4C)alkylenedioxy group, the (1-

4C)alkylenedioxy group is, for example, methylenedioxy -(0CH₂O)- or ethylenedioxy - (OCH₂CH₂O)-.

The process of the invention is particularly suitable for the preparation of a compound of the formula (I⁵), or a pharmaceutically acceptable salt thereof: -A-

(I') wherein each R is as hereinbefore defined; which process comprises hydrolysing a compound of formula (H')

(IP) wherein X and each R is as hereinbefore defined.

According to another aspect, colchinol derivatives of formula (II) may be prepared by reacting, conveniently in the presence of a base, a colchinol derivative of formula (III):

an) wherein each R is as hereinbefore defined; with a phosphorous(V) halide, for example phosphorous oxychloride.

The reaction of the colchinol derivative of formula (III) with the phosphorus (V) halide is suitably carried out in the presence of a base. A suitable base includes, for example an organic base, such as a tertiary amine, for example a tertiary (l-6C)alkylamine such as triethylamine, tributylamine or diisopropylethylamine. A particular base is triethylamine. This step is conveniently carried out in an inert solvent. Suitable inert solvents are those that are unreactive towards phosphorus phosphorous oxychloride, particularly a non- chlorinated organic solvent. More particularly the solvent is selected from a ketone such as methyl isobutyl ketone (MIBK), acetonitrile; and an ether, for example tetrahydrofuran, dioxane or 2-methyltetrahydrofuran. Still more particularly the solvent is an ether such as tetrahydrofuran.

The reaction is suitably carried out at a temperature of from -60 to O⁰C, for example

As will be realised, when the process is used to prepare a compound of the formula (V), the starting material of the formula (II') may be prepared by reacting a colchinol derivative of the formula (III'):

(IIP) wherein each R is as hereinbefore defined; with a phosphorous(V) halide, as hereinbefore defined.

A particular value for R in the compounds of the formulae (I), (I') (II), (II,), (III) and (III') is (l-4C)alkyl, for example methyl.

The process according to this aspect of the invention is particularly suitable for the phosphorylation of N-acetylcolchinol (of the formula (Ilia) defined above), to give ZD6126, or a pharmaceutically acceptable salt thereof. Accordingly in a particular embodiment of the invention there is provided a process for the preparation of ZD6126, or a pharmaceutically acceptable salt thereof which comprises (i) reacting N-acetylcolchinol in the presence of an organic base and an inert organic solvent with a phosphorous (v) halide, for example phosphorous oxychloride (suitably at a temperature below ambient) to give a compound of formula (Ila);

wherein X is halogeno; and (ii) hydrolysing the product of step (i) (suitably in an aqueous medium at a temperature of less than 35⁰C).

According to another particular embodiment there is provided a process for the preparation of ZD6126, or a pharmaceutically acceptable salt thereof which comprises (i) reacting N-acetylcolchinol in the presence of an ether (for example tetrahydrofuran) and a molar excess of triethylamine (relative to the N-acetylcolchinol), with phosphorus oxychloride at a temperature of from -5⁰C to -2O⁰C; and (ii) hydrolysing the product of (i) in an aqueous medium at a temperature below 25⁰C.

The process according to the first aspect of the invention provides phosphorylated colchinol derivatives in high yields and avoids the need to use environmentally undesirable solvents such as chlorinated hydrocarbons. Furthermore, high yields of the desired phosphorylated product are obtained without the need for additional catalysts which can be prohibitively expensive for large scale manufacture.

The phosphorylated colchinol derivative of the formula (I) (such as ZD6126) may be prepared as, or converted into, a pharmaceutically acceptable salt, for example a basic salt such as a calcium, sodium or potassium salt. Salts of a phosphorylated colchinol derivative of the formula (I) may be prepared by conventional means well known in the art, for example by reacting the compound of formula (I) with a metal hydroxide base. Further salts may be prepared using for example conventional salt conversion methods.

The phosphorylated colchinol derivative of the formula (I), prepared according to the process of this first aspect of the invention may be isolated from the resulting reaction mixture by conventional means. However, we have found that certain solvent extraction procedures may be used to provide an aqueous solution of a phosphorylated colchinol derivative of the formula (I) (particularly ZD6126), substantially free of impurities, from which the phosphorylated colchinol derivative of the formula (I) may be isolated in substantially pure form.

Accordingly in an embodiment, the process according to the first aspect of the invention further comprises an extraction step which comprises:

(a) basifying the reaction mixture formed in the hydrolysis process;

(b) isolating the aqueous phase from the organic phase of the resulting mixture;

(c) optionally washing the aqueous phase with a suitable organic solvent;

(d) optionally adjusting the pH of the aqueous phase to approximately 8 to 9 and washing with a suitable organic solvent; and

(e) adjusting the pH to be acidic for example between approximately pH 0 and 1.5 In step (a) suitable base may be added to the reaction mixture to give an alkaline pH for example a pH of from about 12 to about 14. A suitable base includes, for example an inorganic base, potassium hydroxide. Conveniently the base may be added to the reaction mixture as an aqueous solution.

In step (b) aqueous phase is isolated, for example by allowing the aqueous and organic phases to separate and separating the two phases.

In step (c) an optional solvent wash is performed to remove any non-ionised organic impurities that may be present in the aqueous phase. Suitable organic solvents for the wash include, for example an ether such as tetrahydrofuran. Following the wash the aqueous phase is again separated from the organic phase, for example by allowing the two phases to separate.

In Step (d) the pH may be adjusted with any suitable acid, for example hydrochloric acid. A suitable organic solvent for use in optional Step (d) is for example an ether such as tetrahydrofuran. We have found that the optional washing in step (d) effectively removes any residual colchinol derivative of the formula (III) (such as N-acetylcolchinol) which may be present following the phosphorylation reaction. Following step (e) the aqueous solution contains a phosphorylated colchinol derivative of the formula (I), substantially free from impurities, other than inorganic salts. As mentioned hereinbefore the process and subsequent extraction steps are particularly suitable for preparing ZD6126 from N-acetylcolchinol. For the preparation of a pharmaceutical compound in pure form it is generally desirable to be able to obtain a crystalline form of the compound, because crystallisation of the compound can be used as a means for further purification of the compound. We have found that ZD6126 can be crystallised from an aqueous solution under certain conditions. For example crystalline ZD6126 maybe prepared from an aqueous solution of ZD6126 (for example the aqueous solution resulting from the extraction step of the process according to the first aspect of the invention). These crystallisation processes form further aspects of the present invention.

According to a second aspect the invention there is provided a process for isolating ZD6126 as a crystalline solid from an aqueous solution of ZD6126, which process comprises: (i) adjusting the pH of an aqueous solution containing ZD6126 to a value in the range of from about 0 to about 1.5 (for example by addition of a suitable acid, such as a mineral acid such as hydrochloric acid);

(ii) extracting ZD6126 into a suitable organic solvent (for example a solvent selected from an ether such as tetrahydrofuran and acetonitrile, or a mixture thereof, particularly the organic solvent is tetrahydrofuran);

(iii) diluting the organic phase resulting from Step (ii) with water, followed by (iv) removal of solvent (for example by distillation) optionally with additional cycles of water addition, followed by solvent removal, until ZD6126 crystallises.

The crystalline ZD6126 may then be isolated by conventional means, for example filtration and drying.

According to a further aspect of the invention there is provided a process for isolating ZD6126 as a crystalline solid from an organic solution of ZD6126, which process comprises:

(i) diluting an aqueous solution of ZD6126 with a suitable organic solvent (for example a solvent selected from an alcohol, an ether a ketone and a nitrile solvent, particularly a solvent selected from n-butanol, iso-butanol, acetonitrile, tetrahydrofuran, 2- methyl tetrahydrofuran and methyl isobutyl ketone (MIBK), more particularly the solvent is iso-butanol;

(ii) adjusting the pH of the mixture prepared in Step (i) to below about 1.5 (for example by addition of a mineral acid such as hydrochloric acid);

(iii) optionally adding toluene to the mixture followed by removal of water from the mixture (for example by allowing the aqueous and organic phases to separate and removal of the aqueous phase);

(iv) removal of residual water from the organic phase containing the ZD6126 (for example by azeotropic distillation, using either standard distillation methods or using a binary separator or by a combination of both techniques) until the ZD6126 crystallises from the mixture, which is optionally cooled to maximise the amount of crystalline ZD6126 produced.

The crystalline ZD6126 may then be isolated by conventional means, for example filtration and drying. As discussed herein the processes according to the present invention are particularly suitable for the preparation of ZD6126. It may be desirable to crystallise or re-crystallise ZD6126 as a means for further purifying the ZD6126. However, ZD6126 is soluble in very few solvents, for example being the lower alcohols such as methanol and ethanol, and certain solvents in the presence of water, for example water and tertrahyrofuran or water a higher alcohol (such as isopropanol, isobutanol, n-butanol) Once ZD6126 is in solution in hot solvent, the compound does not crystallise readily upon cooling. Furthermore, we have found that attempts to crystallise ZD6126 from aqueous solvents is not a robust process because the ZD6126 is prone to form as an oil in the presence of certain minor impurities. Accordingly, there is a need for an improved method for obtaining ZD6126 in crystalline form. According to another aspect of the present invention there is provided a process for preparing crystalline ZD6126 comprising:

(i) dissolving ZD6126 in a small quantity of methanol (suitably the minimum required to dissolve ZD6126, for example 12 relative volumes of methanol) at an elevated temperature, for example approximately 40°C and optionally screening the solution to remove any un-dissolved ZD6126;

(ii) adding an anti-solvent solvent; (iii) concentrating the resultant mixture (for example by removing solvent by distillation under vacuum at <40°C); (iv) optionally adding a further quantity of anti-solvent; (v) optionally concentrating the resultant mixture (for example by removing solvent by distillation under vacuum at <40°C); (vi) cooling the mixture (for example to about 5°C);and (vii) isolating the crystalline ZD6126 by conventional means (for example by filtration and drying). Suitable anti-solvents which are suitable for use in Step (ii) and optional Step (iv) in this crystallisation process include; isopropanol, isobutanol, n-butanol, methylisobutyl ketone, isopropyl acetate, isopropyl ether. A particular anti-solvent is isopropanol. According to a further aspect of the present invention there is provided a process for preparing crystalline ZD6126 comprising:

(i) dissolving ZD6126 in tetrahydrofuran (for example in 4-6 relative volumes of tetrahydrofuran) containing water (0.3 -1 relative volumes) at elevated temperature, for example 40°C;

(ii) optionally screening the solution to remove any un-dissolved ZD6126;

(iii) adding a small quantity of a suitable anti-solvent (for example 0.5 - 5 relative volumes);

(iv) optionally seeding the mixture with crystals of ZD6126 (0 - 0.5 mol); (v) adding further quantities of the anti-solvent (for example 5 - 25 relative volumes) in aliquots over a period between 1 - 24 hours; (vi) cooling the mixture to between 0 - 10°C; and (viii) isolating the crystalline ZD6126 by conventional means (for example by filtration and drying). Suitable anti-solvents which may be used in this process include isopropyl acetate, isopropanol, isobutyl acetate, n-butyl acetate, isopropyl ether. A particular anti-solvent is isopropyl acetate.

The crystallisation according to the fifth aspect of the invention is particularly suitable for large-scale use (for example 100 L scale or above) because it does not require distillation stages to effect crystallisation of the ZD6126 and is therefore simpler to operate and control. The term "relative volume" used in the above processes refers to the relative amount of solvent used in millilitres relative to the amount of ZD6126 in grams.

According to a further aspect of the present invention there is provided a process for the preparation of ZD6126 which comprises: (i) phosphorylation of N-acetylcolchinol;

(ii) isolating the ZD6126; and

(iii) optionally recrystallising ZD6126 using the process according to the fourth or fifth aspects of the invention.

The phosphorylation in step (i) may be carried out using known methods, for example using the method described in WO 99/02166 (example 1). However, in a particular embodiment phosphorylation is carried out in accordance with the process according to the first aspect of the present invention described hereinbefore. When the ZD6126 is prepared using the phosphorylation process according to the first aspect of the invention, the ZD6126 is suitably isolated using the optional step (iii) of the process according to the first aspect of the invention as hereinbefore described.

Suitable conditions for the optional recrystallisation in step (iii) are as described in relation to the fourth or fifth aspect of the invention.

If required the enantiomerically pure forms of the phosphorylated colchinol derivative of formula (I), such as ZD6126, maybe isolated at any convenient stage of the processes described herein. Suitable methods for the separation of enantiomers are well known. For example the enantiomers may be separated chromatographically using a suitable chiral column; using chiral reagents in combination with NMR spectroscopy; or by derivatisation with a chiral reagent followed by standard achiral separation or spectroscopic techniques.

The invention will now be illustrated in the following non limiting examples, in which standard techniques known to the skilled chemist and techniques analogous to those described in these examples may be used where appropriate, and in which, unless otherwise stated: (i) evaporations were carried out by rotary evaporation in vacuo and work up procedures were carried out after removal of residual solids such as drying agents by filtration; (ii) all reactions were carried out under an inert atmosphere at ambient temperature, typically in the range 18-25⁰C, with solvents technical grade under anhydrous conditions, unless otherwise stated; (iii) the structures of the end products of the formula (I) were generally confirmed by nuclear (generally proton) magnetic resonance (MMR) and mass spectral techniques; magnetic resonance chemical shift values were measured in deuterated dimethyl sulfoxide (unless otherwise stated) on the delta scale (ppm downfield from tetramethylsilane); proton data is quoted unless otherwise stated; spectra were recorded on a on a Bruker DRX500 spectrometer; and peak multiplicities are shown as follows: s, singlet; d, doublet; dd, double doublet; t, triplet; tt, triple triplet; q, quartet; tq, triple quartet; m, multiplet; br, broad; LCMS were recorded on a Waters ZQ Mass Spec Detector, LC column was a SB C8 150mm x 3.0 mm 3.5um (Agilent Zorbax), detection with a HPl 100 with a Diode Array Detector; unless otherwise stated the mass ion quoted is [M + H]⁺; (iv) the following abbreviations may be used hereinbefore or hereinafter:- THF tetrahydrofuran; (v) the term ReI. VoIs (or VoIs) refers to the relative amount of solvent used in millilitres, relative to the amount of the main reaction substrate in grams.

Example 1: Preparation of Crystalline ZD6126 from N-acetvIcolchinol i) Phosphorylation of N-acetylcolchinol

A solution of ZD6126 Phenol (N-acetylcolchinol) (58 g, 134 mmole), in THF (719 ml, <0.05% water), under nitrogen was made-up in a jacketed vessel (vessel A). The temperature of the solution was adjusted to 20°C, and triethylamine (54.5 g, 75.1 ml, 536 ml) was added over 30 min. hi a second vessel (vessel B) phosphorous oxychloride (41.5 g, 25.2 ml, 268 mmole) was added to THF (241 ml) at -18°C (slight exotherm to -20⁰C). When the contents of vessel B had returned to -18⁰C, the contents of vessel A, were added to vessel B at a rate that ensured the temperature did not rise above -15°C (approx. 1.25 hours). THF (48 ml) was added to vessel A as a line wash and then transferred to vessel B. The reaction mixture (vessel B) was then stirred at a temperature between -15°C to — 19°C for 50 minutes, when analysis (HPLC) indicated that there was no remaining N-acetylcolchinol. The mixture was then added to water (480 ml, in vessel C) ensuring that the temperature did not exceed 25°C (approx. 1.25 hours). THF (48 ml) was added to vessel B as a line wash and then transferred to vessel C and the resultant mixture was held at 20°C for 6 hours. An aqueous potassium hydroxide solution (49% w/w) was added to the mixture in vessel C, until the pH was above 13.5 (approx. 128 ml required), at such a rate that the temperature did not rise above 25°C (approximately 1 hour addition time). After stirring for a further 30 minutes at about 2O⁰C, the layers were separated and the upper (organic) layer was discarded. The aqueous layer was recharged to the vessel, THF (480 ml) was added, the mixture was stirred for 30 minutes and the layers were separated. The upper (organic) layer was discarded and the aqueous layer was recharged to the vessel. ii) Isolation by crystallisation from dry isobutanol

Isobutanol (673 ml) was added to the mixture in the vessel, the temperature was adjusted to 15°C (approx.) and the mixture was acidified by the addition hydrochloric acid solution (122 ml, -35% w/w), maintaining the internal temperature below 25°C (addition time approx 45 minutes). The mixture was stirred at 20⁰C for 1 hour, allowed to settle for 20 minutes and then the layers were separated. The lower (aqueous) layer was discarded, water (190 ml) and toluene (190 ml) were added to the remaining solution and the mixture was stirred for 15 minutes, then allowed to settle for 15 minutes. The lower (aqueous) layer was separated and discarded and the vessel was configured for water removal at reflux, under reduced pressure (using Dean-Stark apparatus with a condenser temperature of approx. 1°C). The pressure in the vessel was reduced to approx. 90 niBar and with a j acket temperature of approximately 55°C, distillation occurred at an internal temperature range of 36.5 - 42°C. When approx. 80 ml of water had been removed the batch was a slurry and separation of water had almost ceased. The vessel was then set for reduced pressure distillation and a further 450 ml of solvent were removed at 55-80 mBar. With a jacket temperature of 4O⁰C, the pressure in the vessel was allowed to reach atmospheric and the slurry was cooled to about 5⁰C over 80 minutes, then held at this temperature for 1 hour. The product was filtered under vacuum, washed with isobutanol (2 x 48 ml), then dried in a vacuum oven at 40⁰C, to provide ZD6126, 46.2 g (strength by NMR 99.5%), corrected yield 78.4%; ¹H NMR Spectrum: (D₆ DMSO) 1.88 (3H, s, COMe)₅ 1.89 (IH, m, H-H), 2.04 (IH, m, 10-H), 2.16 (IH, m, H-H), 2.51 (IH, m, 10-H), 3.51 (3H, s, OMe), 3.78 (3H, s, OMe), 3.83, (3H, s, OMe), 4.51 (IH, m, 12-H), 6.79 (IH, s, 1-H), 7.13 (IH, s, 17-H), 7.14 (d, J 8, 19-H), 7.29 (IH, d, J 8, 20-H), 8.41 (lH, br, NH); Mass Spectrum: [MH⁺I 438.

The ZD6126 produced was identical to authentic material by spectroscopic (e.g. ¹H NMR, ¹³C NMR, IR, MS) and chromatographic (HPLC) methods of analysis.

Example 2 Purification of ZD6126 by Recrystallisation From Methanol/ Isopropanol

A solution of ZD6126 (30 g) in methanol (12 relative volumes) was heated to 40⁰C. isopropanol (13 relative volume) was added to the solution and the mixture was concentrated by distillation under reduced pressure (200mbar to 140mbar) at 40⁰C. Further isopropanol (10.0 relative volumes) was added, and the mixture was concentrated by distillation under reduced pressure (100 mbar) at 40°C. The resultant slurry was cooled to 2O⁰C, left overnight, then cooled to 5°C and left for 30 minutes before filtering, washing and drying in a vacuum oven to give crystalline ZD6126. The recovery from the process was 87.7% and the product strength was 99.6% (by HPLC); ¹H NMR Spectrum: (D₆ DMSO) 1.88 (3H, s, COMe), 1.89 (IH, m, H-H), 2.04 (IH, m, 10-H), 2.16 (IH, m, .11-H), 2.51 (IH, m, 10-H), 3.51 (3H, s, OMe), 3.78 (3H, s, OMe), 3.83, (3H, s, OMe), 4.51 (IH, m, 12-H), 6.79 (IH, s, 1-H), 7.13 (IH, s, 17-H), 7.14 (d, J8, 19-H), 7.29 (IH, d, J8, 20-H), 8.41 (IH, br, NH); Mass Spectrum: [MH⁺] 438.

Example 3: Purification of ZD6126 by Recrystallisation from Isopropyl Acetate Tetrahydrofuran (307 mmoles; 25.0 ml; 22.2 g) and water (167 mmoles; 3.00 ml; 3.00 g) was added to a suspension of ZD6126 (11.2 mmoles; 5.00 g), in a 100 ml jacketed vessel, fitted with a temperature probe, and overhead stirrer, was added. Upon heating the mixture to 40°C a solution was obtained, and to this solution was added isopropyl acetate (2.5 ml) in 2 portions. Seed crystals of pure ZD6126 (0.229 mmoles; 100 mg) were then added and the resultant suspension was left to equilibrate for approximately 1 hour, after which time isopropyl acetate (7.5 ml) was added over 4 hours. The mixture was then held overnight, followed by the further additions of isopropyl acetate: (10 ml), over 7.5 hours; followed by (55 ml) over 20 hours. After cooling to 4°C and the mixture was filtered and the filtrate was washed with isopropyl acetate (1 x 5 ml; 1 x 10 ml), then dried under vacuum at 40°C. A white crystalline ZD6126 (4.29g, 98.4% strength [by NMR]) was obtained, representing a recovery 84.4%:1H NMR Spectrum: (D₆ DMSO) 1.88 (3H, s, COMe), 1.89 (IH, m, H-H), 2.04 (IH, m, 10-H), 2.16 (IH, m, H-H), 2.51 (IH, m, 10-H), 3.51 (3H, s, OMe), 3.78 (3H, s, OMe), 3.83, (3H, s, OMe), 4.51 (IH, m, 12-H), 6.79 (IH, s, 1-H), 7.13 (IH, s, 17-H), 7.14 (d, J 8, 19-H), 7.29 (IH, d, J 8, 20-H), 8.41 (IH, br, NH); Mass Spectrum: [MH⁺] 438.

Example 4: Preparation of crystalline ZD6126 and purification by recrystallisation from aquaeous solution

ZD6126 Phenol (23.Og, 64.4 mmol, 1.0 equiv) is dissolved in tetrahydrofuran (189ml, 8.2 vols). Triethylamine (35.9ml, 4.0 equiv) is then added, and this solution is added to a solution of phosphorous oxychloride (12.0ml, 4.0 equiv) in tetrahydrofuran (115ml, 5.0 vol). The resulting dichloridate intermediate reaction mixture is drowned-out into water (230ml, 10 vol), then basified (to pH13) using aqueous potassium hydroxide.

The two phase mixture is separated and the aqueous phase is washed with tetrahydrofuran (230ml, 10 vol), then again at pH8 (after acidification with aqueous hydrochloric acid). Further tetrahydrofuran (230ml, 10 vol) is added, and the solution is acidified to pHl using aqueous hydrochloric acid. The two phase mixture is separated, the organic phase diluted with water (230ml, 10 vol) and the tetrahydrofuran removed by vacuum distillation to precipitate ZD6126 product from aqueous solution. The product (22.9g, 81.3%) is filtered, washed with water and dried under vacuum ¹H-NMR (DMSO): δ 1.86 (m, 2H), 2.02 (m, IH), 2.13 (m, IH), 3.52 (s, 3H), 3.79 (s, 3H)₅ 3.84 (s, 3H), 4.49 (m, IH), 6.79 (s, IH), 7.14 (s, IH), 7.14 (d, IH, J=8.4Hz), 7.30 (d, IH, J=8.1Hz), 8.42 (d, IH, J=8.6Hz). Melting Point : 246.3⁰C

Accurate Mass : 438.1332 (C₂₀H₂₅NO₈P = 438.1318) M ■ 3358, 2992, 2956, 2936, 2863, 2833, 2289 cm^"1. Specific Optical Rotation : [α]_D ²⁵ = -74.5 (c 0.51, MeOH)

Claims

1. A process for the preparation of a phosphorylated colchinol derivative of the formula (I), or a pharmaceutically acceptable salt thereof:

(I) wherein each R, which may be the same or different, is selected from (l-6C)alkyl, benzyl and-C(O)(l-6C)alkyl, or two RO groups together form a (l-4C)alkylenedioxy group; which process comprises hydro lysing a compound of formula (II)

(II) wherein X is halogeno and each R is as hereinbefore defined; and thereafter, if necessary, forming a pharmaceutically acceptable salt thereof.

A process according to claim 1 for preparing a colchinol derivative of formula (Ia)

(Ia) or a pharmaceutically acceptable salt thereof, by hydrolysing a compound of formula (Ha)

(Ha) wherein X is halogeno.

3. A process according to claim 1 or 2 wherein the hydrolysis is performed in an aqueous medium at a temperature of less than 35°C.

4. A process according to any of claims 1 to 3 wherein the compound of formula (II) is prepared by reacting a colchinol derivative of formula (III) or (Ilia):

(III) (Ilia) wherein each R is as defined in claim 1 or 2, with a phosphorous (V) halide.

5. A process according to claim 4 wherein the phosphorus (V) halide is phosphorous oxychloride.

6. A process according to claim 4 or claim 5 wherein the reaction of the colchinol derivative of formula (III) or (Ilia) and the phosphorous (V) halide is carried out in the presence of a base.

7. A process according to claim 6 wherein the base is a tertiary (1-6C) alkylamine. 8. A process according to claim 7 wherein the base is triethylamine.

9. A process according to any of claims 4 to 8 wherein the reaction of the colchinol derivative of formula (III) or (Ilia) and the phosphorous (V) halide is carried out at a temperature of from -3O⁰C to O⁰C.

10. A process according to any of claims 4 to 9 wherein the reaction is carried out in an inert organic solvent selected from methyl isobutyl ketone, acetonitrile, tetrahydrofuran, dioxane or 2-methyltetrahydrofuran.

11. A process according to claim 10 wherein the solvent is tetrahydrofuran.

12. A process according to claim 2 wherein the hydrolysis is carried out in an aqueous medium at a temperature below 25⁰C and wherein the compound of formula (Ha) is prepared by reacting N-acetylcolchinol with phosphorous oxychloride in the presence of an ether and a molar excess of triethylamine and at a temperature in the range of -5⁰C to -2O⁰C.

13. A process according to any preceding claim comprising the further step of extracting the colchinol derivative of formula (I) as an aqueous solution.

14. A process according to claim 13 wherein the method for extracting the colchinol derivative of formula (I) comprises the steps of:-

(a) adding a base to the reaction mixture formed in the process of any of claims 1 to 12; (b) isolating the aqueous phase from the organic phase of the mixture resulting from step (a);

(c) optionally washing the aqueous phase from step (b) with a suitable organic solvent;

(d) optionally adjusting the pH of the aqueous phase to a pH in the range of pH 8 to 9 and washing with a suitable organic solvent; and (e) adjusting the pH of the resulting aqueous phase to a pH in the range of pH 0 to pH 1.5.

15. A process according to claim 14 wherein in step (a) the base is added to give a pH in the range of pH 12-ρH 14. 16. A process according to claim 15 wherein the base is potassium hydroxide.

17. A process according to any of claims 14 to 16 wherein wash step (c ), if present, is performed using tetrahydrofuran.

18. A process for preparing a compound of formula (Ia), as hereinabove defined, in the form of a crystalline solid , which process comprises the steps of:-

(i) adjusting the pH of an aqueous solution containing a compound of formula (Ia) to a value in the range of from pH 0 to pHl .5 ;

(ii) extracting the compound into a suitable organic solvent;

(iii) diluting the resulting organic phase with water ;

(iv) removing the organic solvent; and

(v) repeating steps (iii) and (iv) ,as necessary, until the compound crystallises.

19. A process according to claim 18 wherein in step (i) the pH of the aqueous solution is adjusted by addition of hydrochloric acid.

20. A process according to claim 18 or 19 wherein the organic solvent used in step (ii) is selected from tetrahydrofuran and acetonitrile, or a mixture thereof.

21. A process for preparing a compound of formula (Ia) ,as hereinabove defined, in the form of a crystalline solid , which process comprises the steps of:-

(i) diluting an aqueous solution of formula (Ia) with a suitable organic solvent; (ii) adjusting the pH of the mixture prepared in step (i) to below 1.5 ;

(iii) optionally, adding toluene to the resulting mixture followed by removal of water from the mixture; and

(iv) removing residual water from the organic phase containing the compound of formula (Ia) until the compound of formula (Ia) crystallises from the mixture. 22. A process according to claim 21 wherein the organic solvent in step (i) is selected from n- butanol, iso-butanol, acetonitrile, tetrahydrofuran, 2-methyl tetrahydrofuran and methyl isobutyl ketone.

5 23. A process according to claim 22 wherein the solvent is iso-butanol

24. A process for preparing a compound of formula (Ia), as hereinbefore defined, in crystalline form comprising the steps of:-

(i) dissolving the compound of formula (Ia) in methanol at an elevated temperature and 10 optionally screening the resulting solution to remove any un-dissolved compound;

(ii) adding an anti-solvent solvent;

(iii) concentrating the resultant mixture;

(iv) optionally adding a further quantity of anti-solvent;

(v) optionally concentrating the resultant mixture;

15 (vi) cooling the mixture;and

(vii) isolating the crystalline product.

25. A process according to claim 24 wherein step (i) is carried out at a temperature of approximately 40 ⁰ C.

20

26. A process according to claim 24 or claim 25 wherein the anti-solvent used in step(ii) and in step (iv), if present, is selected from isopropanol, isobutanol, n-butanol, methylisobutyl ketone, isopropyl acetate, isopropyl ether.

25 27. A process for preparing a compound of formula (Ia), as hereinbefore defined, in crystalline form comprising the steps of:-

(i) dissolving the compound of formula (Ia) in tetrahydrofuran containing water at elevated temperature;

(ii) optionally screening the resulting solution to remove any un-dissolved compound of 30 formula (Ia);

(iii) adding an anti-solvent ;

(iv) optionally seeding the resulting mixture with crystals of compound of formula (Ia); (v) adding further quantities of the anti-solvent in aliquots over a period between 1 - 24 hours;

(vi) cooling the mixture to between 0 - 10°C; and (viii) isolating the crystalline product.

28. A process according to claim 27 wherein the anti-solvent used is selected from isopropyl acetate, isopropanol, isobutyl acetate, n-butyl acetate, isopropyl ether.