HRP930515A2 - New nucleosides and processes for their preparation - Google Patents

Description

The invention relates to new pyrimidine nucleosides that can be used as anti-tumor agents, both to 5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine themselves, as well as to their physiologically tolerable addition salts of acids and their production, as well as pharmaceutical preparations based on these new compounds and their production.

The compounds according to the invention can be easily produced starting from 5-fluorocytidine or 5-fluorouridine in the same way as 5'-deoxycytidine or 5'-deoxyuridine is produced from cytidine or uridine. Thus, the initial nucleosides can, for example, be halogenated in the 5' position by a known procedure, directly or, what is better, after prior protection of both hydroxy groups in the 2'- and 3'-positions by forming the appropriate ketal. The resulting 5'-halogen compounds are then reduced to the corresponding 5'-halogen compounds either by means of catalytic hydrogenation or by means of reducing agents, such as metal hydrides. In the case of a non-ketalized halogen compound, the desired final product is obtained directly. If the hydroxyl groups were previously ketalized, then the protective group is removed after the reduction by means of hydrolysis in the usual way and thus the desired final product is obtained. Finally, 5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorocytidine can, if desired, be converted into physiologically tolerable acid addition salts in a known manner by reaction with a physiologically tolerable acid.

The term "physiologically tolerable salts" includes non-toxic salts that 5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine form in the reaction with inorganic mineral acids and organic acids, for example hydrochlorides, hydrobromides, phosphates, sulfates, nitrates, acetates, formates, maleates, fumarates or benzoates.

Presumed protecting groups with 2',3'-hydroxy groups are alkylidene, cycloalkylidene and aralkylide groups, which can be further substituted. Examples of such protecting groups are anisilidene, cyclohexylidene, methoxymethylidene and, in particular, isopropylidene or benzylidene.

The introduction of 5-fluorocytidine or 5-fluorouridine into a 2'.3'-ketal can be carried out in a known manner. Thus, the fluoronucleoside can be reacted in a particularly favorable way with an organic sulfonic acid, for example p-toluenesulfonic acid, and a ketalizing agent, for example 2,2-dimethoxy-propane in a favorable organic solvent, preferably a ketone such as acetone, at a temperature of 0° to 60°C, preferably room temperature.

The introduction of a halogen atom in the 5'-position can be realized either in 5-fluorocytidine or 5-fluorouridine or, which is the best, in a corresponding 2'.3'-ketal. As halogen, it is best to take bromine and iodine. The introduction of iodine can, for example, be carried out by reaction with an iodinizing agent such as methyltriphenoxyphosphonium iodide (MTPI) in a polar aprotic organic solvent such as dimethylformamide (DMF) at a temperature between 0° and 100°C, best at room temperature. The introduction of bromine can be carried out by reaction with a brominating agent such as triphenyl-phosphine/tetra-bromo-carbon, in a polar aprotic solvent such as DMF, at a temperature between 10° and 100°C.

The conversion of the 5'-halo-compound to the corresponding 5'-deoxy-compound can easily be carried out by means of catalytic hydrogenation in a protic polar solvent such as an alcohol, preferably methanol, with the use of a noble metal catalyst such as palladium, in a given case on to some inert carrier material such as coal or barium sulfate or, also, in the presence of nickel. Hydrogenation can be carried out at a temperature between 0 and 60°C, preferably at room temperature and under a pressure of 1 to 5 atmospheres, preferably at normal pressure, in the presence of an organic base, preferably a tri-lower alkyl-amine such as triethyl- Amen.

Other suitable reducing agents for the production of the 5'-deoxy compound from the corresponding 5'-halogen compound are complex metal hydrides such as tributyl tin hydride, sodium cyano boron hydride or lithium - triethyl boron hydride. The reaction with these agents can be carried out at temperatures between 0 and 100°C in the presence of solvents known for these purposes.

Removal of the ketal group in the 2',3'-position of the nucleoside can be easily achieved by hydrolysis, in already known ways. Thus, for example, the isopropylidene group can be removed at room temperature by the action of trifluoroacetic acid.

Intermediate products 5'-deoxy-2',3'-isopropylidene-5-fluorocytidine and 5'-deoxy-2',3'-0-isopropylidene-5-fluorouridine, which, according to the invention, are obtained using an isopropylidene protecting group, are new compounds and, also, the subject of this application.

5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine as well as their physiologically tolerable acid salts are active against Ehrlich carcinoma and sarcoma 180 in mice within a wide range of oral and parenteral dosages. These compounds are, therefore, valuable agents against tumors and can be used as drugs in the form of pharmaceutical preparations with direct or delayed release of the active substance. In mixtures with non-toxic, inert, solid or liquid carriers such as, for example, water, gelatin, gum arabic, milk sugar, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols, petroleum jelly, etc., which are favorable for the central (on example oral) or parenteral application. Pharmaceutical preparations can be in solid form, for example as tablets, dragees, suppositories, capsules, or in liquid form, for example as solutions, suspensions or emulsions. In certain cases, they are sterilized, that is, they contain other auxiliary substances such as preservatives, stabilizers, cross-linkers or emulsifiers, flavor enhancers, salts to change the osmotic pressure or buffers. The production of pharmaceutical preparations can be carried out in a manner that is routine for any expert.

Anti-tumor tests:

For administration to animals, the compounds are dissolved in water.

Sarcoma 180-test:

18 - 20 g white mice were implanted using a trocar with small tumor parts (20 - 30 mg) subcutaneously in the right groin area. Tumor sections were taken from animals bearing tumors implanted 7-10 days earlier. Treatment was started on the day of implantation and continued once a day for 8 days. Animals were killed 8 days after implantation and tumors were removed and measured. The ratio of the average tumor weight of the untreated control group (C) to the average tumor weight of the treated group (T) was calculated. Inhibition of tumor growth is given in percentage as (C-T) x 100/C. A compound was considered active for a given dose if growth inhibition was 50 percent or more.

Ehrlich's carcinoma-test:

The solid form of this tumor was obtained by subcutaneous implantation of 0.5 ml of an ascitic tumor cell suspension diluted 1-10 with table salt solution. A white mouse weighing 18-20 kg, which had a tumor implanted 7-10 days earlier, served as a donor. The treatment and presentation of the results was carried out in the manner described earlier.

The results obtained with the compounds described by this invention as well as with two compounds of today's practice are shown in tables 1 and 2.

Table 1:

Activity of pyridine nucleosides against sarcoma 180 in mice

[image] Table 2:

Action of primidine nucleosides against Erling's carcinoma in mice

[image]

Examples

Example 1

A suspension of 5-fluorocytidine (92 g) and ptoluenesulfonic acid monohydrate (80 g) in acetone (1500 ml) and 2,2-dimethoxy-propane (200 ml) was stirred for 2 hours at room temperature. It is then added to the excess sodium bicarbonate and mixed until the acid is neutralized. The solid residue was drained and washed with acetone, and the filtrate and washing solution were evaporated to dryness. The residue was treated with hot ethyl acetate (700 ml) and after cooling, its crystallization began slowly. After standing overnight, the solid residue was washed with ethyl acetate and dried in a vacuum. Yield: 99.5 g (94 %) of 2',3'-O-isopropylidene-5-fluorocytidine. One sample was recrystallized from a mixture of methanol/ethyl acetate: m.p. 182 - 184°C.

A solution of 2',3'-O-isopropylidene-5-fluorocytidine (32 g) and methyl-triphenoxy-phosphonium-iodide (MTPI, 60 g) in dimethylformamide (DMF, 300 ml dried) was allowed to stand for 1 1/2 hour at room temperature. Then methanol (100 ml) is added to it and after 30 minutes the mixture is evaporated to an oily consistency. Then, 700 ml of ethyl acetate and 700 ml of 5% aqueous sodium thiophosphate are added and, after shaking, the layers are left to separate. The ethyl acetate layer was washed once with aqueous thiosulfate solution (700 ml) and twice with water (700 ml) and evaporated to an oily residue which was dissolved in hot ethyl acetate (400 ml). Hexane is added to the hot solution until crystallization begins. After standing at 0°C, the crystalline precipitate is washed with hexane and dried in a vacuum. After processing and mother liquor, a total of 30.1 g (69%) of 5'-deoxy-5'-iodo-2',3'-O-isopropylidene-5-fluorocytidine was obtained, m.p. 192 - 194°C.

A solution of 5'-deoxy-5'-iodo-2'-O-isopropylidene-5-fluorocytidine (48g) in methanol (500ml) and triethylamine (20ml) was hydrogenated at room temperature and under normal pressure for 30 minutes in in the presence of palladium on charcoal (5%, 25 g) with constant stirring. The catalyst was removed by filtration over Celite®, the filtrate was evaporated to dryness and ethyl acetate (200 ml) was added to the residue. It was left to stand overnight, then the crystals were drained, the filtrate was evaporated to about 100 ml and once again left to stand overnight. The crystals formed again were drained, and the filtrate was evaporated to dryness under vacuum. 31 g (93%) of 5'-deoxy-2',3'-O-isopropylidene-5-fluorocytidine was obtained in the form of a foam. This substance is characterized by the formation of crystalline picrate, m.p. 168 - 170°C.

5'-deoxy-2',3'-O-isopropylidene-5-fluorocytidine (31 g) was treated with 90% trifluoroacetic acid (200 ml) for 40 minutes. The solution was evaporated to dryness, and the residue, in order to remove water and trifluoroacetic acid, was repeatedly evaporated with ethanol and finally dissolved in ethyl acetate (400 ml). The solution was made alkaline with triethylamine and after a few minutes crystallization began. After standing overnight, the crystals were washed with ethyl acetate and dried in a vacuum. 14 g (49%) of 5'-deoxy-5-fluorocytidine were obtained. An additional amount was obtained by chromatography of the mother liquor on a silica gel column (600 g), where the elution was performed with ethyl acetate (4 L) as well as with a mixture of ethyl acetate/methanol (5:1, by volume, 4 L). Fractions of interest were evaporated to dryness and run over a Dowex 50® column (H+-form 2.3 x 60 cm). Then the column was washed, first with water and finally with ammonia solution (1N). The ammonia fractions were evaporated to dryness and the residue was recrystallized from methanol. In this way, another 6.7 g of 5'-deoxy-5-fluorocytidine was obtained. Total yield: 20.7 g (78%); d.p. 209 - 211°C.

Example 2

5'-deoxy-5'-iodo-5-fluorocytidine (1.5 g) in methanol (30 ml) and triethylamine (1 ml) was hydrogenated with constant stirring for 90 minutes at room temperature and normal pressure in the presence palladium on charcoal (0.75 g, 10%). The catalyst was separated by filtration, washed with methanol, the filtrate was evaporated to dryness and then dissolved in water (100 ml). The aqueous solution was passed through a column of Dowex 50® (H+-form, 2.3 x 30 cm). It was first eluted with water (1 L), then with aqueous ammonium hydroxide (2N, 2 L). The ammonia eluates were evaporated to dryness and the residue was extracted twice with 200 ml each of ethanol and evaporated and finally recrystallized from ethanol. 0.685 g (69%) of 5'-deoxy-5-fluorocytidine m.p. were obtained. 207 -208°C. The starting substance was obtained in both ways described below:

a) from 5-fluorocytidine

A solution of 5-fluorocytidine (2.61 g) in DMF (50 ml) was treated with MTPI (5.42 g) for 5 hours at room temperature. Since the presence of the starting reagent could still be proven in the reaction mixture by means of thin layer chromatography, MTPI (5.42 g) was added once more. After 90 minutes, 10 ml of methanol was added and after 15 minutes the solution was evaporated to an oily consistency. The residue was dissolved in a mixture of ethyl acetate/methanol (1:1, volume ratio, 30 ml) and transferred to a silica gel column (600 g). The column was eluted using ethyl acetate/methanol (10:1, volume ratio). Fractions of 20 ml each were collected. Fractions 190-280 were combined, evaporated, and the solid residue was dissolved in water (30 ml). The aqueous solution was applied to a Dowex 50® column (H+-form 2.3 x 40 cm). It was eluted first with water and then with 2 N ammonium hydroxide. The eluate is

evaporated to a crystalline mass weighing 200 mg (5.4 %). Recrystallization from ethanol gave a pure substance m.p. 187 - 189°C.

b) from 5'-deoxy-5'-iodo-2'.3'-O-isopropylidene-5-fluorocytidine

A solution of 5'-deoxy-5'-iodo-2',3'-O-isopropylidene-5-fluorocytidine (20 g) in a mixture of trifluoroacetic acid and water (9:1, parts by volume, 100 ml) was kept at at room temperature for 70 minutes and then evaporated to dryness. The residue was dissolved twice in 200 ml of ethanol and the solution was evaporated each time, and the residue was finally dissolved in ethyl acetate (200 ml). It was then neutralized with triethylamine and after standing overnight the crystalline precipitate was dried. Yield: 16.8 g (93%).

Example 3

A suspension of 5-fluorouridine (50 g) and p-toluenesulfonic acid monohydrate (39.3 g) in acetone (750 ml) and 2,2-dimethoxy-propane (94 ml) was stirred at room temperature for 50 minutes. An excess of solid sodium carbonate was added to the clear solution and the mixture was stirred until it was neutral. The solid residue was removed by filtration, washed with acetone and the filtrate was evaporated to dryness. The solid residue was recrystallized from ethyl acetate (2 L) to give 48 g (83 %) of 2',3'-O-isopropylidene-5-fluorouridine, m.p. 196 - 197°C.

A solution of 2',3'-O-isopropylidene-5-fluorouridine (46.4 g) in DMF (250 ml, dry) was kept at room temperature for 50 minutes in contact with MTPI (86.7 g). Ethanol (200 ml) was then added and after 30 minutes the solution was evaporated, and the oily residue was partitioned between ethyl acetate (1 L) and aqueous sodium thiosulfate (5%, 1 L). The ethyl acetate layer was washed twice with 1 L of water each, dried with sodium sulfate and evaporated to dryness. The oily residue was crystallized from ethyl acetate (350 ml). 52.9 g (85%) of 5'-deoxy-5'-iodo-2',3'-0-isopropylidene-5-fluorouridine were obtained, m.p. 202 - 203.5°C.

A solution of 5'-deoxy-5'-iodo-2',3'-O-isopropylidene-5-fluorouridine (24 g) in methanol (800 ml) and triethylamine (15 ml) was hydrogenated for 90 minutes at room temperature, under normal pressure, with constant stirring in the presence of palladium on charcoal (12 g, 5%). The crystallizer was drained over celite® and washed with methanol, while the filtrate was evaporated to dryness, and 200 ml of ethyl acetate was added to the residue and left to stand for 1 hour. The crystalline precipitate was removed by filtration, the filtrate was evaporated to half and allowed to stand overnight and filtered once more. The resulting filtrate was evaporated to dryness in a vacuum and thus obtained 5'-deoxy-2',3'-O-isopropylidene-5-fluorocytidine /UV(CH3OH) : λ.max 204 mm (ε 10900) and 267 mm (ε 8670). IR (Kbr) : 3380, 3200, 1710, 1670 [image] [image] /, which was further treated with dilute trifluoroacetic acid (90%, 200 ml) for 1 hour. The product was evaporated to dryness and to remove water and trifluoroacetic acid, ethanol was repeatedly added and the solution evaporated again, and the residue was crystallized from ethyl acetate. 11.35 g of 5'-deoxy-5-fluorouridine were obtained, m.p. 189 - 190°C.

Example 4

A solution of 5'-deoxy-5'-iodo-5-fluorouridine (291 mg) in methanol (10 ml) and triethylamine (0.5 ml) was hydrogenated at room temperature and under normal pressure in the presence of palladium on charcoal (5 %, 145 mg) 1 1/2 hours. The catalyst was drained, the filtrate was evaporated to dryness and the residue was dissolved in a minimum amount of hot ethyl acetate. During cooling, triethylammonium iodide crystallized, which was removed by straining. The filtrate was evaporated to dryness and recrystallized from ethanol. 130 mg (68%) of 5'-deoxy-5-fluorouridine was obtained.

The starting substance was obtained in both of the following ways:

a) from 5-fluorouridine

A solution of 5-fluorouridine (2.62 g) in DMF (50 mL) was treated with MTPI (5.42 g) 1 1/3 gas at room temperature. 30 minutes after the addition of 10 ml of methanol, the solution was evaporated to an oily residue which was dissolved in ethyl acetate (30 ml) and transferred to a silica gel column (500 g). The column was eluted with ethyl acetate, collecting fractions of 20 ml each. Fractions 61-130 were pooled, evaporated to dryness and the residue was dissolved in hot ethyl acetate (50 ml). After adding hexane (10 ml), a crystalline substance was obtained. After standing overnight at room temperature, the crystalline precipitate was washed with hexane and dried under reduced pressure. Subsequent processing and mother liquor yielded a total of 1.13 g (30%) of 5'-deoxy-5'-iodo-5-fluorouridine m.p. 174.5-175.5°C.

b) from 5'-deoxy-5'-iodo-2'.3'-O-isopropylidene-5-fluorouridine

4 g of 5'-deoxy-5'-iodo-2',3'-O-isopropylidene-5-fluorouridine was treated at room temperature for 15 minutes with a mixture of trifluoroacetic acid and water (9:1, parts by volume, 30 ml ). The solution was evaporated to dryness, 100 ml of ethanol was added twice and evaporated, and the residue was recrystallized from ethyl acetate. Yield: 1.865 g (88 %).

Example 5

Tablets, which contain one piece

mg mg

[image] 329.0 655.5

Production is carried out by mixing components 1-4, granulating with water, drying overnight and grinding. Components 5 and 6 were mixed afterwards. Mixing was done for 5 minutes and the mixture was pressed into tablets.

Example 6

Tablets containing per piece

[image]

mg mg

327.5 655.0

The production was carried out by mixing components 1, 3 and 4 in one mixer, granulating with component 2 in alcohol, drying overnight and grinding. Then magnesium stearate was added to the granulate and the mixture was pressed into tablets.

Example 7

Capsules containing one piece

[image]

Production:

Components 1 and 2 were mixed for 10 minutes in a mixer, then components 3 and 4 were added. It was mixed for 5 minutes and filling was done on a suitable machine.

Example 8

(a) 5 g of 5'-deoxy-5-fluorocytidine, or 5'-deoxy-5-fluorouridine, was dissolved in 75 ml of distilled water. The solution was subjected to bacteriological straining and distributed in 10 ampoules under sterile conditions. Freeze drying was then carried out, so that each ampoule contained 500 mg of the active substance.

(b) 500 mg each of 5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine in the form of pure crystals were placed in ampoules that were heated and sterilized by heating.

Before use, the dry preparations are reconstituted for parenteral administration by adding an appropriate aqueous solution, such as water for injections, isotonic saline or 5% dextrose solution.

Claims (8)

1. Process for the production of 5'-deoxy-5-fluorocytidine, i.e. 5'-deoxy-5-fluorouridine as well as their physiologically tolerable acid addition salts, indicated by the fact that the 2',3'-O-protecting group in 2' ,3'-positions of ketalized 5'-deoxy-5-fluorocytidine or 5'-deoxy-5-fluorouridine is broken off hydrolytically, or that 5'-deoxy-5'-halo-5-fluorocytidine, or 5'-deoxy-5 '-halo-5-fluorocytidine reduction in position 5' and that in the desired cases, the resulting 5'-deoxy-5-fluorocytidine or 5'-deoxy-5-fluorocytidine is treated with a physiologically tolerable acid. 2. The method according to claim 1, characterized in that the 2',3'-O- protecting group is an alkylidene, cyclo-alkylidene- or aracylidene-group which can be further substituted. 3. The method according to claim 1, characterized in that the 2'3'-O- protecting group is an isopropylidene group. 4. The method according to claim 1, characterized in that 5'-deoxy-2',3'-O-isopropylidene-5-fluorocytidine or 5'-deoxy-2',3'-O-isopropylidene-5-fluorouridine are hydrolyzed using trifluoroacetic acid. 5. The method according to claim 1, characterized in that 5'-deoxy-5'-halo-5-fluorocytidine or 5'-deoxy-5'-halo-5-fluorouridine is catalytically hydrogenated. 6. The method according to claim 1, characterized in that 5'-deoxy-5'-halo-5-fluorocytidine or 5'-deoxy-5'-halo-5-fluorouridine are reduced using a complex metal hydride. 7. The method according to claim 1, characterized in that 5'-deoxy-5'-iodo-5-fluorocytidine or 5'-deoxy-5'-halo-5-fluorocytidine are reduced. 8. Process for the production of pharmaceutical preparations, indicated by the fact that 5'-deoxy-5-fluorocytidine, i.e. 5'-deoxy-5-fluorouridine or some of their physiologically tolerable addition salts with acid, as an active substance, are mixed with the usual solid or liquid carrier material suitable for therapeutic administration, non-toxic and inert, which is usual for such preparations and that the resulting mixture is brought into a favorable galenic form.