HK1161237B - Process for the preparation of calcobutrol - Google Patents

Description

Method for preparing combretastatin calcium

Technical Field

The present invention relates to a process for the preparation of calcium complexes of 10- (2, 3-dihydroxy-1- (hydroxymethyl) propyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triacetic acid, also known as cobutralcium, and their use for the preparation of galenic formulations. The invention further relates to cobutrol having a purity level not disclosed so far.

Background

Combretastatin is an additive in galenic formulations of gadobutrol, which solves the problem of preventing the release of free gadolinium in the formulation (solution). Gadobutrol is a gadolinium-containing contrast agent for nuclear spin tomography, which has been approved in Germany as Gadovist since 2000For indications "contrast enhancement of skull and spinal Magnetic Resonance Tomography (MRT)" (EP0448181B1, EP0643705B1, EP0986548B1, EP0596586B1 and CA patent 1341176). Gadobutrol is a nonionic complex composed of gadolinium (iii) and the macrocyclic ligand 10- (2, 3-dihydroxy-1- (hydroxymethyl) propyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triacetic acid (butrol). Gadovist is sold as a 1 molar aqueous solution having the following components in the formulation: gadobutrol, calcium salt of combretazole, tromethamine, hydrochloric acid and water for injection.

It has been found to be advantageous to apply an excess of complex-forming ligand in the form of a calcium complex in the formulation for most gadolinium-containing contrast agents (EP0270483B 2). The calcium complex acts to prevent the release of free gadolinium from the formulation (e.g. by storage for years, re-complexing with foreign ions in the glass).

The synthesis of the calcium complex (combretastatin) is described in detail in Inorg. chem.1997, 36, 6086-. However, none of the processes disclosed therein provides cobutrol with the purity required by authorities. The exact replication (reproduction) method of route 3 (p. 6088-6089) gives a substance with a purity of only 94%, as determined by HPLC (stationary phase: Hypersil Phenyl (5 μm) from SHANDON; mobile phase: acetonitrile/borate buffer (pH 8) at a volume ratio of 20/100; detection: UV detector (200 nm); injection volume: 10. mu.l). The available ligand from the synthesis of gadobutrol (butrol) does not have the high purity required to transfer it directly to the calcium complex. Further purification is difficult due to the zwitterionic nature of the ligand. Unlike the ligands BOPTA, DTPA and DOTA (see US5595714) which crystallize at pH values of 1.7-1.8, it is not possible to crystallize butrol at any pH (see comparative examples below) and thus to purify it by crystallization. Without being bound by a particular theory, the difference in crystallization capacity is due to the dihydroxy-hydroxymethyl-propyl side chain in butrol, which is not present in BOPTA, DTPA or DOTA. The lack of crystallization may be due to differences in polarity or differences in the ability to form hydrogen bonds. Finally, another possible reason may be the so-called "glycerol effect" from the dihydroxy-hydroxymethyl-propyl side chain, i.e. the ability of glycerol to prevent water crystallization at 0 ℃, which breaks hydrogen bonds in the crystal.

Although neutral gadolinium complexes (gadobutrol) can be purified in ion exchange columns (e.g. Amberlite IRC50, Amberlite FPC3500, or Amberlite IRA 67) and subsequently obtained in very high purity (more than 99.7%) by very efficient crystallization (e.g. from ethanol, preferably ethanol with less than 200ppm water), this is not possible with cobutrcalcium (because of the additional acid functionality). Purification of the calcium complex was unsuccessful because even by preparative HPLC, there were still impurities that were very close to the main peak that could not be separated. Several different methods of separation of cobutrol by HPLC have been tried (changing mobile phase, gradient, etc.), but none of them achieved separation.

In Ca²⁺In the presence of (25 ℃, in 0.1N KCl) with different Ca²⁺: ligand ratio, the thermodynamic stability constant and acid dissociation constant of combretastatin have been determined by pH-potential equilibrium titration (pH-potentiometric equilibrium) of the ligand (butrol). The results were:

Log(K_CaL-)＝14.67±0.02 K_CaL-＝[CaL^-]/[Ca²⁺][L^3-]

pK_a＝3.39±0.12 K_a＝[CaL^-][H⁺]/[CaLH]

based on these measurements, the distribution of calcium between free calcium ions, neutral complexes (combretastatin, ligands having two negative charges) and anionic complexes (ligands having three negative charges) for different pH values can be calculated. The results are shown in FIG. 1. It is clear that at any pH value, the neutral complex is not higher than 20% in the calcareous mass (species). Preparative methods in aqueous solutions may result in some impurities due to the equilibrium between the calcium-containing species.

Although the anionic complex is the predominant species at higher pH values, this is not very useful for purification purposes. Salts with this complex (e.g. sodium salts) are not suitable for the work-up. The sodium salt of the complex is a highly hygroscopic, glassy substance that cannot be handled on any practical scale. Thus, in the preparation of the Gadovist solution, the sodium salt is prepared in situ by adding sodium hydroxide to the cobutrcalcium.

The large difference in stability between gadobutrol and combretastatin is the primary reason for making combretastatin useful in Gadovist formulations, i.e. the large difference in stability between the gadolinium complex and the calcium complex means that the calcium complex will scavenge any free gadolinium ions by forming a gadolinium complex.

The object of the present invention is to obtain very pure combretastatin, preferably in crystalline form, in as high a yield as possible.

Drawings

Figure 1 shows the distribution of calcium-containing species with pH for cobutrone calcium complexes and related compounds.

Detailed Description

It was surprisingly found that starting from very pure gadobutrol, it is possible to prepare combretastatin efficiently. Removing gadolinium from gadobutrol complex by decomplexation to obtain a ligand with high purity, then combining said ligand with Ca²⁺And (4) complexing.

Decomplexation of gadolinium complexes with oxalic acid by addition of mineral acids, preferably hydrochloric acid, has been described in the literature for ligands other than butrol. It is disclosed in US5595714 how to recover gadolinium and free ligand from gadolinium containing contrast agents by decomplexation with oxalic acid/hydrochloric acid. US5595714 does not disclose the use of a process for preparing calcium salts.

The present invention relates to a process for the preparation of a calcium complex of 10- (2, 3-dihydroxy-1- (hydroxymethyl) propyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triacetic acid (combretastatin), wherein:

a) decomplexation of the gadolinium complex of 10- (2, 3-dihydroxy-1- (hydroxymethyl) propyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triacetic acid (gadobutrol) by a decomplexer,

b) the precipitated gadolinium salt is removed and,

c) binding the free ligands in the solution obtained from step b) to an acidic ion exchange resin,

d) eluting the resin with an aqueous alkaline solution,

e) treating the eluate with an acidic ion exchange resin, and

f) reacting the ligand with Ca²⁺Complexing and crystallizing.

The term "decomplexer" in the present invention refers to an agent capable of forming a Gd salt which is only slightly soluble in water. Examples of decomplexers are oxalate ion sources such as oxalic acid and phosphate ion sources such as phosphoric acid, which form insoluble gadolinium oxalate and gadolinium phosphate salts, respectively. Preferred decomplexers are oxalic acid and phosphoric acid, most preferably oxalic acid.

Decomplexation is particularly preferably carried out in water at a temperature of from 75 to 100 deg.C, for example from 80 to 95 deg.C, such as from 87.5 to 92.5 deg.C, preferably about 90 deg.C.

After liberation of the ligand (butrol), it is treated with an acidic ion exchange resin, especially at a pH of 3.65 to 3.80, preferably about 3.72. Examples of useful ion exchange resins are Amberlite 252C or Amberlite IR 120.

The preferred basic aqueous reagent for the elution is a base which can be removed from the aqueous solution by distillation. This reagent has the advantage that it will be removed from the ligand-containing eluate by evaporation of water. The basic aqueous reagent may be ammonia or a volatile amine. Herein, the term "volatile amine" refers to any aliphatic primary, secondary or tertiary amine having 1 to 4 carbon atoms independently in the alkyl chain attached to the central nitrogen atom and which has a boiling point at atmospheric pressure of 95 ℃ or less, such as 80 ℃ or less, 70 ℃ or less, 60 ℃ or less, 50 ℃ or less, 40 ℃ or less, preferably 30 ℃ or less, or 15 ℃ or less. Examples of volatile amines are methylamine, ethylamine, n-propylamine, isopropylamine, dimethylamine, diethylamine, triethylamine, trimethylamine, di-n-propylamine, diisopropylamine, n-butylamine, sec-butylamine, 1-amino-2, 2-dimethylpropane, 2-amino-2-methylbutane, 2-amino-3-methylbutane, 2-aminopentane and 3-aminopentane. In a preferred embodiment of the present invention, the basic reagent used for the elution is ammonia, dimethylamine, methylamine, ethylamine, trimethylamine, isopropylamine or a mixture thereof, more preferably ammonia, dimethylamine or a mixture thereof, and most preferably ammonia.

In one embodiment, the free ligand is directly bound to Ca after treatment with the ion exchange resin, i.e., without prior separation of the free ligand²⁺And (4) complexing. In another embodiment, the reaction is carried out with Ca²⁺Prior to complexation, the free ligand is isolated by freeze-drying.

Calcium carbonate, calcium oxide or calcium hydroxide are preferred Ca for complexation²⁺A source of ions. The complexation is preferably carried out in aqueous solution at a temperature of from 75 to 100 deg.C, for example from 80 to 95 deg.C, such as from 87.5 to 92.5 deg.C, preferably about 90 deg.C.

It has surprisingly been found that the addition of a mineral acid as described in US5595714 is not necessary for decomplexing. If high-purity gadobutrol is reacted directly in water with a stoichiometric amount of decomplexer, for example oxalic acid, a high-quality and purity colorless aqueous solution of butrol is obtained after quantitative decomplexation and filtration of the gadolinium oxalate. Thus, in one embodiment, the decomplexer, such as oxalic acid, is added to gadobutrol at a pH higher than 2, for example at a pH higher than 3, such as a pH higher than 4, preferably a pH higher than 4.5. The combretazole calcium complex can be directly prepared from a solution of butrol after further purification.

To ensure that no free gadolinium is present in the butrol solution, it is subjected to an ion exchange treatment. To remove any remaining ions, the butrol solution was loaded onto an ion exchange column and washed thoroughly with water. Subsequently, the ligand is eluted with an aqueous basic solution such as aqueous ammonia, and the aqueous eluate is gently evaporated under vacuum. The residue was diluted with water and, after treatment with activated carbon, the pH was adjusted to 3.7 by addition of an acidic ion exchange resin. The exchange resin was filtered off and the solution was subsequently freeze-dried.

Phosphoric acid may be used instead of oxalic acid. In this case, gadolinium phosphate (GdPO)₄) And (4) precipitating. The ligands can be worked up in a similar manner.

In principle, the complexation with the calcium salt can be continued and carried out directly. However, it has been found that the ligand can be isolated by mild freeze-drying, in this way giving a convenient storage form.

The final reaction to form cobutrol is carried out by complexing butrol with a stoichiometric amount of calcium carbonate in water under heating. However, CaO and Ca (OH) can also be used₂。

To remove particles and nuclei, treatment with activated carbon and subsequent filtration was used. The filtrate was evaporated under vacuum as much as possible and then crystallized by addition of ethanol. For this purpose, a heating reflux and subsequent cooling are carried out. The precipitated crystalline product was filtered and washed with a small amount of ethanol. Then, it was dried at 70 ℃ in a vacuum vessel. It has been found that crystallisation can also be achieved from acetone or isopropanol, however the preferred solvent is ethanol.

Without being bound by a particular theory, it has been found that crystallization of combretastatin in ethanol or other suitable solvent drives the equilibrium (as shown in figure 1) between free calcium ions and ligands in aqueous solution (on the one hand) and combretastatin complex (on the other hand) towards a stable crystalline complex. Thus, with the stoichiometric amounts of calcium ions and butrol at the beginning of the complexation step, only the complex remains after crystallization. However, it was also found that in order to form pure combretastatin, pure butrol must be employed in the complexation step. If the butrol is impure, combutra calcium will also contain similar levels of impurities. This was observed according to the method of Inorg. chem.1997, 36, 6086-.

In one embodiment, the invention relates to a process in which 10- (2, 3-dihydroxy-1- (hydroxymethyl) propyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triacetic acid gadolinium complex (gadobutrol) is decomplexed with oxalic acid or phosphoric acid in water under heating, the precipitated gadolinium oxalate/phosphate is filtered off, the free ligand is bound to an acidic ion exchange resin, the resin is eluted with an aqueous ammonia solution, the solution is evaporated and then the acidic ion exchange resin is usedAdjusting the pH to 3.6-3.8, freeze drying the solution, and heating the ligand and Ca²⁺Ion complexation, crystallization of the complex from ethanol after complete reaction, and drying of the crystals under vacuum after isolation.

The cobutrol calcium prepared in this way is characterized by a very high quality. The product was colorless and soluble in water and had a purity of 99.0% or more, in some batches 99.4% or more (purity according to HPLC, 100% method). The whole process from gadobutrol to combretastatin is characterized by high reproducibility and operability. An overall yield of 91.2% is very satisfactory. The product is storage stable and can be used to formulate Gadovist solutions. The sodium salt of cobutrol was obtained in situ by the addition of stoichiometric amounts of sodium hydroxide. The Gadovist solution prepared in this way is stable for years and provides safety, i.e. toxic gadolinium is never released into the solution.

This enables the provision of cobutrol with high purity at low cost, which can be used directly for further processing and preparation of Gadovist, meeting the expectations of authorities and physicians.

The invention also relates to the use of calcium complexes of 10- (2, 3-dihydroxy-1- (hydroxymethyl) propyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triacetic acid for the preparation of commercial galenic formulations of gadobutrol.

Examples

HPLC, 100% method

Stationary phase: hypersil ODS, 3 μm, or equivalent packaging material

125×4.6mm

Mobile phase:

eluent A: an aqueous solution having 2.0g of sodium octane sulfonate monohydrate per liter was prepared.

Adjusting pH to 2.0 + -0.1 with sulfuric acid

Eluent B: acetonitrile for chromatography

Gradiometer:

time [ min ]]	Eluent A [ vol%]	Eluent B [ vol%]
			0	87	13
20	87	13
			45	70	30
50	70	30
			51	87	13
60	87	13

Flow rate: 1.0ml/min

UV detection wavelength: 197nm

Sample concentration: 7mg/1ml of eluent A

Injection volume: 10 μ L

Comparative example

It is not possible to prepare cobutrol by the method disclosed in US 5595714. Gadobutrol and hydrochloric acid are then stirred with oxalic acid in water (pH 0.8) at 20 ℃ for about 6h, and the precipitated gadolinium oxalate is filtered off, as described in this us patent. The filtrate was divided into several portions and the pH was adjusted by adding 20% aqueous sodium hydroxide solution. Since it is not known at which pH the ligand crystallizes, several solutions each having a pH difference of 0.1 were tested. The results of the crystallization experiments are provided in the following table:

pH value	After 24 hours	After 1 week	After 1 month
				0.8	Non-crystal	Non-crystal	Non-crystal
0.9	Non-crystal	Non-crystal	Non-crystal
				1.0	Non-crystal	Non-crystal	Non-crystal
1.1	Non-crystal	Non-crystal	Non-crystal
				1.2	Non-crystal	Non-crystal	Non-crystal
1.3	Non-crystal	Non-crystal	Non-crystal
				1.4	Non-crystal	Non-crystal	Non-crystal
1.5	Non-crystal	Non-crystal	Non-crystal
				1.6	Non-crystal	Non-crystal	Non-crystal
1.7	Non-crystal	Non-crystal	Non-crystal
				1.8	Non-crystal	Non-crystal	Non-crystal
1.9	Non-crystal	Non-crystal	Non-crystal
				2.0	Non-crystal	Non-crystal	Non-crystal
2.1	Non-crystal	Non-crystal	Non-crystal
				2.2	Non-crystal	Non-crystal	Non-crystal
2.3	Non-crystal	Non-crystal	Non-crystal
				2.4	Non-crystal	Non-crystal	Non-crystal
2.5	Non-crystal	Non-crystal	Non-crystal
				2.6	Non-crystal	Non-crystal	Non-crystal
2.7	Non-crystal	Non-crystal	Non-crystal
				2.8	Non-crystal	Non-crystal	Non-crystal

2.9	Non-crystal	Non-crystal	Non-crystal
				3.0	Non-crystal	Non-crystal	Non-crystal
3.1	Non-crystal	Non-crystal	Non-crystal
				3.2	Non-crystal	Non-crystal	Non-crystal
3.3	Non-crystal	Non-crystal	Non-crystal
				3.4	Non-crystal	Non-crystal	Non-crystal
3.5	Non-crystal	Non-crystal	Non-crystal
				3.6	Non-crystal	Non-crystal	Non-crystal
3.7	Non-crystal	Non-crystal	Non-crystal
				3.8	Non-crystal	Non-crystal	Non-crystal
3.9	Non-crystal	Non-crystal	Non-crystal
				4.0	Non-crystal	Non-crystal	Non-crystal

Attempts to crystallize the ligand according to the teachings of said us patent were unsuccessful. HPLC studies showed that the appearance of new impurities was observed under strongly acidic conditions, which resulted in a decrease in the quality of the ligand. Moreover, it was observed that the originally colorless ligand turned yellow after 6h and the impurities could not be removed. The above-described process from said us patent is not suitable for the preparation of cobutrol, since further work-up of the ligand only results in cobutrol with a colored purity of 93% (HPLC, 100% process).

However, it has surprisingly been shown that isolation via crystallization is not necessary. It has also been found that it is possible to remove gadolinium from the complex so that the highly pure ligand in solution can be reacted directly or isolated by freeze-drying under mild conditions.

Example 1: preparation of butrol

26.255kg of gadobutrol (water content 4.78%, purity > 99% ensured by ion exchange and crystallization) and 10.108kg of oxalic acid dihydrate (solid, 99%) were poured into a vessel with stirrer, 175 l of deionized water were added and the mixture was stirred at 90 ℃ for 5 hours. The mixture was cooled to 20 ℃ (pH measurement gave a value of 3.1-3.5). The precipitated gadolinium oxalate was aspirated and washed twice with 50l of water. The filtrate was loaded onto a cation exchange column packed with 250l of Amberlite 252C, and the column was then washed with water.

The product was eluted from the column with a mixture of 250l of deionized water and 125l of 25% aqueous ammonia and collected in 13 portions.

Fraction of eluate	Volume (l)	pH	TLC discovery1
				1	50	4.4	No products
2	50	4.6	No products
				3	45	4.4	No products
4	45	3.9	No products
				5	20	3.6	Product of
6	30	3.7	Product of
				7	40	3.7	Product of
8	25	4.9	Product of
				9	20	5.4	Product of
10	45	10.9	Product of
				11	50	12.2	No products
12	50	12.3	No products
				13	100	2.3	No products

The 5-10 th eluate is evaporated in vacuo on a rotary evaporator at a bath temperature of 70 ℃ to approximately 35 l. The pH of the concentrate was 6.7.

The oily residue was dissolved in 125l of deionized water and 3.75kg of activated carbon (NoritSX PLUS, previously rinsed thoroughly with water) was added, and the solution was then heated to an internal temperature of 90 ℃ for 1 hour. The warm solution was filtered to remove the activated charcoal and the charcoal was washed 3 times with 25l of water each time at 70 ℃.

The mixture was cooled to 20 ℃ and the pH was adjusted to 3.72 by addition of acidic ion exchange resin (Amberlite IR 120, resin added in portions, 6.5l each, for a total of 45.5 l; 100ml of probe (probe) was used-additional resin did not change the pH). The ion exchange resin was filtered off and washed 4 times with 25l portions of deionized water. The filtrate together with the washing water was evaporated in vacuo at 70 ℃ in a heated stirrer to a volume of nearly 100 l. The solution was cooled to 20 ℃ and then freeze-dried in a freeze-dryer.

Yield: 18.15kg (17.69 kg: 95% of theory, adjusted for water) of a colorless amorphous powder

Water content (Karl-Fischer): 2.60 percent

Elemental analysis (corrected for water):

element(s)	C	H	N	O
					Theoretical value	47.99	7.61	12.44	31.96
Measured value	47.77	7.73	12.38	32.04

HPLC purity (100% method): more than 99 percent

Example 2: preparation of combretazole calcium

A total of 3.356kg of calcium carbonate (99.3%) was added in portions to 15.39kg of butrol (water content: 2.6%) dissolved in 120l of deionized water, and stirred at an internal temperature of 90 ℃ for 1 hour. It was then cooled to 20 ℃ and 1.5kg of activated carbon (Norit SX PLUS; the carbon was previously washed thoroughly with water) was added. Stirred at 20 ℃ for 1 hour and then the charcoal was filtered off. The charcoal was washed three times with 15l of water each time.

Subsequently, the filtrate together with the washing water was evaporated in vacuo to an oil at 80 ℃ in a heated stirrer, which still could be stirred and corresponded to 1.4 times the original butrol. 150l of ethanol were added to the oil, which was then boiled under reflux for 3 hours. Cooled to 20 ℃ and the precipitated crystal suspension is filtered off. The crystals were washed twice with 15l of ethanol each time.

The still moist product obtained from ethanol was dried in a vacuum dryer set at 70 ℃ until the weight was constant.

Yield: 16.27kg (96% of theory) of colourless crystals

Elemental analysis:

element(s)	C	H	N	O	Ca
						Theoretical value	44.34	6.41	11.49	29.53	8.22
Measured value	44.54	6.57	11.34	29.43	8.17

HPLC purity (100% method): more than 99.0 percent

Experiments corresponding to this example were carried out on a laboratory scale using acetone and isopropanol as solvents for the crystallization instead of ethanol. Similar purities were obtained.

Claims (8)

1. A process for the preparation of a calcium complex of 10- (2, 3-dihydroxy-1- (hydroxymethyl) propyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triacetic acid (cobutrcalcium), wherein:

a) decomplexation of the gadolinium complex of 10- (2, 3-dihydroxy-1- (hydroxymethyl) propyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triacetic acid (gadobutrol) by a decomplexer,

b) the precipitated gadolinium salt is removed and,

c) binding the free ligands in the solution obtained from step b) to an acidic ion exchange resin,

d) eluting the resin with an aqueous alkaline solution,

e) treating the eluate with an acidic ion exchange resin, and

f) reacting the ligand with Ca²⁺Complexing and crystallizing the mixture to obtain a complex,

wherein:

the decomplexer in step a) is an oxalate ion source or a phosphate ion source,

the decomplexation reaction of step a) is carried out in water at a temperature of 75-100 ℃,

the pH value in step a) is higher than 2 before the addition of the decomplexing agent, and

the aqueous alkaline solution in step d) is an aqueous solution of a base, which can be removed from the aqueous solution by distillation.

2. The process of claim 1, wherein the decomplexing agent in step a) is oxalic acid.

3. The process of claim 1, wherein the aqueous alkaline solution in step d) is a solution of ammonia, a volatile amine or a mixture thereof.

4. The process of claim 3, wherein the basic aqueous solution in step d) is a solution of ammonia, dimethylamine, methylamine, ethylamine, trimethylamine, isopropylamine or a mixture thereof.

5. The process of claim 4, wherein the aqueous alkaline solution in step d) is an ammonia solution.

6. The process of any one of claims 1-5, wherein the product obtained from step e) is isolated by freeze-drying.

7. The process of any one of claims 1 to 5, wherein the product obtained from step e) is reacted directly with a source of calcium ions without prior isolation.

8. The process of any one of claims 1 to 5, wherein the source of calcium ions in step f) is calcium carbonate, calcium oxide or calcium hydroxide.