BG60540B2 - Nucleosides and method for their preparation - Google Patents

Abstract

The invention relates to new pyrimidinenucleosides, especially 5'-desoxy-5-fluorocytidine and 5'-desoxy-5-fluororidine used as antitumour preparations, to their physiologically acceptable, adjuctive salts with acids, to a method for their preparation and to pharmaceutical preparations produced based on these compounds.

Description

The invention relates to novel pyrimidinucleosides, in particular to 5'-deoxy-5-fluorocytidine and 5'-deoxy-5fluorouridine, which are used as antitumor agents, as well as to their physiologically acceptable acid addition salts and to a method for their preparation , as well as pharmaceutical preparations containing these novel compounds.

The compounds of the invention can be readily prepared from 5-fluorocytidine or 5-fluorouridine, in particular by methods analogous to the methods for preparing 5'-deoxycytidine and 5'-deoxyuridine from cytidine and uridine, respectively. Thus, for example, the starting nucleosides can be halogenated in a known manner at the 5-site, directly or after preliminary protection of the two hydroxyl groups of the 2'- and 3-site by the formation of ketals. The resulting 5 halogenated compounds are then converted into the corresponding 5'-deoxy compounds by reduction, either by catalytic hydrogenation or by using reducing agents, such as e.g. complex metal hydrides. In case the hydroxyl groups are not ketalized _? the desired compound is obtained directly. When the hydroxyl groups are pre-catalysed, then after reduction the protecting groups are cleaved by hydrolysis in a known manner to obtain the desired final product. may be converted to physiologically acceptable acid addition salts by reacting in a known manner with physiologically acceptable acid.

The term "physiologically acceptable salts" includes non-toxic salts obtained by reacting 5'-deoxy5-fluorocytidine and 5-deoxy-5-fluorouridine being inorganic mineral acids, as well as with organic acids, e.g. hydrochloride, hydrobromide, phosphate, sulfate, nitrate, acetate, formate, maleate, fumarate or benzoate

Preferably the 2 ', 3'-hydroxy-protecting groups are alkylidene, cycloalkylidene, aralkylidene groups which may further contain substituents. Examples of such protecting groups are anisylidene, cyclohexylidene. methoxymethylidene and preferably isopropylidene or benzylidene groups.

The conversion of 5-fluorocytidine or 5-fluorouridine to 2 ', 3-ketal can be accomplished in a known manner. For example, in a preferred way of effecting this conversion, the fluoronucleoside is reacted with an organic acid, e.g., p-toluenesulfonic acid, and with a catalysing agent, e.g. 2,2-dimethyloxypropane, in a suitable organic solvent, preferably a ketone, such as e.g. acetone at a temperature of about 0 ° C to 60 ° C, preferably at room temperature.

The introduction of halogen atoms into the 5'-position can be carried out as in 5-fluorocytidine, respectively in 5-fluorouridine, or even preferably in 2 ', 3'-ketal. Bromine and iodine are preferred as halogen atoms. Introducing iodine may • l

- ο take place eg. by reacting 1, a iodizing agent such as methyltriphenoxyphosphonium iodide in a polar aprotic organic solvent, e.g. dimethylformamide, at a temperature in the range from 0 to 100 ° C, preferably at room temperature. The introduction of bromine can be accomplished by reacting a brominating agent such as triphenylphosphine / carbon tetrabromide in a polar aprotic organic solvent such as dimethylformamide at a temperature between 10 and 100 C.

The conversion of the 5'-halogenated compound to the corresponding 5-deoxy compound can be easily carried out by catalytic hydrogenation in a protogenic polar solvent, e.g. alcohol, preferably methanol, using catalysts containing noble metals such as palladium, optionally on an inert carrier, eg carbon or barium sulphate, or also in the presence of nickel. The hydrogenation may take place at a temperature in the range of from 0 to 60 ° C, preferably at room temperature and at a pressure of 1 to 5 atmospheres, preferably at normal pressure, in the presence of an organic base, preferably tri-lower alkylamine such as triethylamine.

Other suitable reducing agents for the preparation of 5'deoxy compounds from the corresponding 5'-halogenated compounds are complex metal hydrides such as tributyltin hydride. sodium cyanoborohydride or lithium triethyl borohydride. The reduction with these agents can be carried out at temperatures from 0 to 100 ° using the solvents known for carrying out this reaction.

Cleavage of the ketal groups at the 2 ', 3'-position of the nucleoside can be easily accomplished by hydrolysis of known

- 4 special way. For example, an isopropylidene group may be cleaved by treatment with trifluoroacetic acid at room temperature.

If using an isopropylidene protecting group is obtained! according to the invention the intermediates 5-deoxy and -2 '. 3 '- O - and z ο π ρ ο p i l i d e n - 5 - f l u ο ρ o t i t and d n n and 5'-deoxy-2', 3'-Oisopronylidene-5- fluorouridine, which are new compounds.

5 '- d e s o k s and - 5 - f l u ο ρ o ts and t and d and n u t and 5' - d e s o k s and - 5 - f l y ο ρ o u r and day, as well as their physiologically acceptable acid addition salts, have activity against Ehrlichcarcinoma and sarcoma 180 in mice with oral and parenteral administration over a rather wide dosage range. These compounds are also valuable antitumor agents and can be used as pharmaceuticals in the form of pharmaceuticals for direct or delayed administration of the active substance in appropriate mixtures with substances intended, e.g. for enteral (e.g. oral) or parenteral administration Suitable substances for use in these mixtures are non-toxic inert solid or liquid carriers, such as e.g. water, gelatin, arabic gum, milk sugar, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols. petroleum jelly, etc. pharmaceutical preparations may be prepared in solid form such as tablets, dragees, suppositories, capsules, or in liquid form, e.g. such as solutions, suspensions, emulsions. In some cases, they are subjected to sterilization or the like, or contain adjuvants such as preservatives, stabilizers, crosslinkers, emulsifiers, flavoring agents, salts that alter osmotic pressure or buffers. The preparation of the pharmaceutical preparations can be carried out in any manner known to those skilled in the art.

Attempts to test for tumor activity1:

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

Experience with sarcoma 180:

18-20 g of white mice were implanted with a subcutaneous tumor (20-30 mg) in the right side of the groin area. Tumorige fragments are removed from the animals after they have been worn 7 to 10 days after implantation. On the day of implantation, the animals are treated with the test substances administered once daily for S days. Eight days after the pranet o implant is treated, the animals are killed, the tumors are excised and weighed. Calculate the ratio of the average weight of tumors in the untreated group (control group C) to the mean weight of tumors in the treated group (T) The retention in tumor development, expressed as a percentage, is obtained when calculating the following dependence: ( C - T) 100 / C. A compound at a given dose is considered active when the calculated retention in% is -50.

Experiments with Ehrlich carcinoma:

A solid form of this tumor is obtained by subcutaneous implantation of 0.5 ml of a solution of ascites tumor cells diluted from 1 to 10 spots with sodium chloride solution. The experiments were performed on white mice weighing 18-20 g, in which the tumor was implanted 7 to 10 days in advance. The procedure and evaluation of the results were performed as described above.

- 6 The results of ic obtained using compounds of the invention as well as those compounds known in the art are summarized in the following Table 1 and Table 2 and Table 2

Table 1

Effect of pyrimidinucleosides against sarcoma 180 in mice with

_______5 '- d e h ο k s i - 5 - fluorocytes di n__5'-deoxy-5-fl uorouri d e n

Dose x 8 experienced survived- stuck- experienced survived- stuck- mice led Jeanne [%] mice led jane [%] 400 i.p. 16 14 95 200 16 14 93 16 15 88 100 16 16 86 16 16 88 50 16 15 72 16 15 89 · - 16 16 64 16 14 67 12.5 8 8 37 8 7 43 400 years 16 16 92 16 15 90 200 16 13 82 16 15 90 100 16 14 76 16 16 83 50 16 16 65 16 16 80 25 16 15 76 16 16 75 12.5 24 23 62 16 16 73 6.25 8 7 26 15 15 68 3.12 16 16 51 1.56 8 8 30

Effect of pyrimidinucleosides against Ehrlich carcinoma in mice

Table 2

1 Compound dose x 8 experienced yarn - ? and dr - I 1 mice led jane [%] 5'-D is zoxy-5- 400 i.p 15 14 Q1 fluorocytidine 200 24 23 72 100 16 16 65 1 50 24 23 57 25 15 15 ⁴⁵ 800 years 8 8 99 1 400 16 1 < ⁹⁵ i 200 24 24 80 100 24 24 ⁷¹ 1 50 16 16 58! 1 I 1____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ 25 16 16 37 5-D Esoxy-5- 400 i.p. 16 14 98; 1 fluorouridine 200 24 22 86 100 24 20 71 1 50 24 23 59 ί 1 25 24 22 43 i 800 years 8 8 99 400 16 16 ⁹⁸ 200 16 16 90 I 1 100 15 15 70! 50 8 8 56? 25 8 8 27 .. j

Compound dose x 8 experienced p r e w - stuck- L ^P1 EW 1______ m and w to and led w a n e 5'-D is about okridine 200 i.p. 8 8 9 100 8 8 41 2 ', 5'-Dideoxy-5- 400 i.p. 16 14 81 fluorouridine 200 16 14 68 100 16 14 37 200 years 8 8 34

(

The following examples illustrate the invention

Example 1

A suspension of 5-fluorocytidine (92 g) and p-toluenesulfonic acid monohydrate (80 g) in acetone (1500 ml) and 2,2 dimethoxypropane (200 ml) was stirred for 2 hours at room temperature. Solid sodium bicarbonate was added in excess and the reaction mixture was stirred to neutralize the acid. The solid was removed by filtration and washed with acetone. The filtrate and washing liquid were concentrated to dryness. The residue was treated with hot ethyl acetate (700 ml) and slowly began to crystallize. After being left overnight _? the resulting solid was filtered off, washed with ethyl acetate and dried in vacuo. Yield of 99.5 g (94%) of 2 ', 3'-isopropylidene-5-fluorocytidine is obtained. A sample of this product was crystallized from a methanol / ethyl acetate mixture and had a liquefaction point of 182-184 ° C.

A solution of 2 ', 3'-O-isopropylidene-5-fluorocytidine (32 g) and methyltriphenoxyphosphonium iodide (60g) in dimethylformamide (300 ml, dry) was maintained for one and a half hours at room temperature. Methanol (100 µl) was then added and after 3 minutes the reaction mixture was concentrated to an oily consistency and partitioned with ethyl acetate (700 ml) and aqueous sodium thiosulfate (5%, 700 ml). The ethyl acetate layer was washed once with aqueous thiosulphate (700 ml) and twice with water (700 ml) and evaporated to give an oil which was dissolved in hot ethyl acetate (400 ml). Hexane was added to the hot solution until crystallization began. After leaving the reaction mixture at 0 ° C, the crystallized precipitate was separated, washed with hexane and dried in vacuo. The mother liquors were treated to extract an additional amount of product and a total of 30.1 g (69%) of 5'-deoxy-5'-iodine-2 ', 3'-O-isopropylidene-5-fluorocytidine was obtained with a liquid point of 192 -194 ° C.

A solution of 5'-deoxy-5'-iodine-2 ', 3'-O-isopropylidene-5-fluorocytidine (48 g) in methanol (500 ml) and triethylamine (20 ml) was hydrogenated with constant stirring at room temperature and normal pressure for 30 minutes in the presence of palladium on charcoal (5%, 25 g). The catalyst was removed by filtration through celite, the filtrate was evaporated to dryness and treated with ethyl acetate (200 ml). The mixture was allowed to stand overnight, the crystals formed were separated by filtration and the filtrate was evaporated to a volume of 100 ml and again allowed to stand overnight. The crystals formed were filtered again and the filtrate was evaporated to dryness under reduced pressure. 31 g (93%) of 5'-deoxy-2 ', 3'-O-isopropylidene-5-fluorocytidine are obtained as a foam. This material is characterized by the preparation of its crystalline picrate salt having a liquid point of 168-170 ° C.

5- Deoxy-2'.3 '- () - isopropylidene-5-fluorocarbons ι and d and (31 g) was treated for 40 minutes with 90% solution of 1 Rfluoroacetic acid (200 ml ). The solution was evaporated to dryness, the residue was repeatedly treated with ethanol and evaporated to remove residues from water and trifluoroacetic acid and finally dissolved in ethyl acetate (400 ml). The mixture was left overnight and the separated crystals were washed with ethyl acetate and dried in vacuo. 14 g (49%) of 5'-deoxy-5-fluorocytidine are obtained. An additional amount of the product was obtained by chromatography on a mother liquor through a silica gel column (600 g) eluting with ethyl acetate (4 L) and ethyl acetate / meanol (in a volume ratio of 5: 1, 4 I). The fractions thus obtained were evaporated to dryness and passed through a column of Dowex 50 (H ⁺ form, 2.3 x 60 cm) _? which is washed with water and then with ammonia (1N). The ammonia fractions were evaporated to dryness and the residue was recrystallized from methanol This gave an additional 6.7 g of 5'-deoxy-5-fluorocytidine. Total yield: 20.7 g (78%) with a liquefaction point of 209-211 ° C.

Example 2

5'-deoxy-5'-iodine-5-fluorocytidine (1.5 g) in methanol (30 ml) and triethylamine (1 ml) was hydrogenated with constant stirring at room temperature and normal pressure for 90 minutes in the presence of palladium on charcoal (10%, 0.75 g). The catalyst was removed by filtration and washed with methanol, the filtrate evaporated to dryness and dissolved in water (100 ml). The aqueous solution was passed through a column of Dowex 50 (H ⁺ form, 2.3 x 30 cm), eluting with water (11) and then with ammonium hydroxide (2N, 2L). Ammonium-alkali eluates

- 11 steaming to dryness, the axis is treated twice with 200 nil of ethanol, subjected to evaporation and finally the product crystallizes from ethanol. Obtained O.6S5 g (69%) of 5'-deoxy5-fluorocytidine with a liquefaction point of 2O7-2O8 ° C

The starting product is obtained in one of the following two ways:

a) from 5-fluorocytidine

A solution of 5-fluorocytidine (2 61 g) in dimethylformamide (50 ml) was treated with methyltriphenoxyphosphonium iodide (5.42 g) for 5 hours at room temperature. As it was found by thin layer chromatography that unreacted starting material was present in the reaction mixture, methyltriphenoxyphosphonium iodide (5.42 g) was added once more to the mixture. After 90 minutes, 10 ml of methanol was added and 15 minutes after that the solution was evaporated to give an oily consistency. The residue was dissolved in a mixture of ethyl acetate and methanol (in a volume ratio of 1.1, 30ml) and passed through a silica gel column (600 g). The column was eluted with ethyl acetate / methanol (10: 1 by volume). 20 ml fractions were collected. fractions 190-280 were combined and evaporated to give a solid residue which was dissolved in water (30 ml). The aqueous solution was passed through a column of Dowex 50 (H ⁺ form, 2.3 x 40 cm) which was eluted with water and then with a solution of ammonium hydroxide (2N). The eluate was evaporated to give a crystalline mass of 200 mg (5.4%). Recrystallization from ethanol gave pure material with a melting point of 187-189 ° C.

b) from 5'-deoxy-5'-iodine-2 ', 3'-0-isopropylidene-5-fluorocytidine

Times ί in op at 5 - de zo and 5 '- iodine - 2'. The 3-O-isopropyl and ί is n-5 - φ.ι> oro-pytidine (20 g) in trifluoroacetic acid water (9: 1, 100 ml by volume) is kept at room temperature for After 70 minutes, then evaporated to dryness, the residue was treated twice with portions of but 200 ml of ethanol, which were evaporated and then dissolved in ethyl acetate (200 ml). The solution was neutralized with triethylamine and allowed to crystallize overnight. Obtained 16.S g (93%) of the product.

χ. Example 3

A suspension of 5-fluorouridine (50 g) and p-toluenesulfonic acid monohydrate (39.3 g) in acetone (750 ml) and 2,1 dimethoxyproyan (94 ml) was stirred for 50 minutes at room temperature. sodium bicarbonate in excess and the reaction mixture was stirred to neutralize the acid. The solid was removed by filtration and washed with acetone, and the filtrate and washing liquid were concentrated to dryness. The residue was recrystallized from ethyl acetate (2 L). A yield of 48 g (83%) of 2 ', 3'-O-isopropylidene-5fluorouridine was obtained with a liquid point of 196-197 ° C.

A solution of 2 ', 3'-O-isopropylidene-5-fluorouridine (46 4 g) p dimethylformamide (250 ml, dry) was treated with methyltriphenoxyphosphonium iodide (86.7 g) and kept at room temperature for 50 minutes. Ethanol (200 ml) was then added and after 30 minutes the reaction mixture was concentrated to give an oily consistency and partitioned by treatment with ethyl acetate (1 L) and aqueous sodium thiosulfate (5%, 1 L). The ethyl acetate layer was washed twice with water (1 L), dried (Na 2 SO 4) and evaporated to a half-wait oil, which was triturated and boiled in ellate ia i (350 ml) from which crystallized 5 2 0 g (85 %) 5'-deoxy-5'-iodine-2 ', 3'-Oisoiroylidene-5-fluorouridine with a liquefaction point of 202203.5 ° 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 with constant stirring at room temperature and normal pressure for 90 minutes in the presence of palladium on charcoal (5%, 12 g). The catalyst was removed by filtration through celite and washed with methanol. until the filtrate was evaporated to dryness and treated with ethyl acetate (200 ml) for one hour. The crystals formed were separated by filtration and the filtrate was evaporated until its volume was halved and allowed to stand again overnight. The crystals formed were filtered again and the filtrate was evaporated to dryness under reduced pressure. Prepared 5'-deoxy-2 ', 3'-O-isopropylidene-5-fluorouridine (which was characterized by UV spectrum (CH 3 OH):? _Max 204 mm .- (ε 10900) and 267 mm (a S670) and IR spectrum (KBr): 3380,3200, 1710,1670 cm @ -1) and treated for 1 hour with aqueous trifluoroacetic acid (90%, 200 ml). The product was evaporated to dryness, the residue was repeatedly treated with ethanol and evaporated to remove residual water and trifluoroacetic acid and finally recrystallized from ethyl acetate. 11.35 g of 5'-deoxy-5-fluorouridine are obtained with a liquid point of 189-190 ° C.

Example 4

A solution of 5'-deoxy-5'-iodine-5-fluorouridine (291 mg) in methanol (10 ml) and triethylamine (0.5 ml) was hydrogenated for one and a half hours in the presence of palladium on valle (5 145 mg) ) at room temperature and under normal pressure The catalyst is removed by filtration and the filtrate is evaporated to dryness and the residue is dissolved in a minimum amount of hot ethyl acetate. Upon cooling the solution crystallizes triethylammonium iodide, which is removed by filtration and the filtrate is evaporated to dryness and recrystallized from ethanol. 130 mg (68%) of 5'-deoxy-5fluorouridine are obtained.

The starting products are obtained by the following two methods:

a) from 5-fluorouridine

A solution of 5-fluorouridine (2.62 g) in dimethylformamide (50 ml) was treated with methyltriphenoxyphosphonium iodide (5.42 g) for 75 minutes at room temperature. 10 ml of methanol was added and 30 minutes thereafter the solution was evaporated to give an oil which was dissolved in ethyl acetate (30 ml) and purified over a silica gel column (500 g). The column was eluted with ethyl acetate, collecting fractions of up to 20 ml. Fractions 61 to 130 were combined, evaporated to dryness and the residue was dissolved in hot ethyl acetate (50 ml). Crystalline material was obtained after the addition of hexane (10 ml). The mixture was allowed to stand overnight at room temperature, the crystalline product obtained was separated, washed with hexane and dried under reduced pressure. Treatment of the mother liquors gave a total of 1.13 g (30%) of 5'-deoxy-5'iodo-5-fluorouridine with a melting point of 174.5-175.5 ° C.

b) from 5'-deoxy-5'-iodine-2 ', 3'-O-isopropylidene-5-fluorouridine g 5'-deoxy-5'-iodine-2', 3'-0-isopropylidene- The 5-fluorouridine is treated with trifluoroacetic acid / water (9: 1, 30 ml by volume) at room temperature for 15 minutes and then evaporated to dryness, the residue is treated twice with portions of oi per JOO ml eiano.1, κοπιο evaporates! after which the product was recrystallized from ethyl acetate to afford a yield of 1.S65 g (88%).

Example 5

Composition of one tablet

Ί mg 1. 5'-deoxy-5-fluorocytidine or __________- 5'-deoxy-5-fluorouridine 250 500 2 Modified starch 25 50 3. Gelatinized starch 25 50 4. Corn starch 25 50 5. Stearic acid 2.5 2.5 6. Magnesium stearate 1.5 3.5 ^! 329.0 655.5

The tablets are prepared by mixing components 1 to 4 with water, drying the mixture overnight and then grinding. The components 5 and 6 are then added and after stirring for 5 minutes the tablets are compressed.

Example 6

Composition of one tablet

mg mg 1. 5'-deoxy-5-fluorocytidine or 5'-d ezoxy-5-fluorouridine 250 500 2. Corn starch 50 50 3. Magnesium stearate 2 5 4. Talc 10 20 312 575

The tablets are prepared by mixing components 1. h and 4 in a mixer, granulating is a solution of component 2 in alcohol, then the mixture is dried overnight and milled. Then magnesium sgearate is added to the granules and the mixture is pressed.

Example Ί

Composition of one capsule

mg mg 1. 5'-deoxy-5-fluorocytidine or 5 ' - ezoxy-5-fluorouridine 250 500 2. Polyvinylpyrrolidine 25 50 3. Modified starch 25 50 4. Cornstarch 25 50 5. Magnesium stearate 2.5 5.0 327.5 655.5

The capsules are prepared by stirring components 1 and 2 for 10 minutes in a mixer, then adding components 3 and 4. The mixture is stirred for 5 minutes and the capsules are filled with this mixture using a suitable device.

Example 8

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

* 7 ό) 500 mg or 5'-deoxy-5-flu> Iroids I indine or cboiBemo 5'-deoxy-5-fluorouridia in the form of pure crystals is filled into ampoules to be soldered! and sterilized by heating

Gsi dry detergents used! by adding suitable water-containing solvents such as water for injection, isotonic sodium chloride solution or dextrose solution for parenteral administration.

Claims (6)

Π A Τ E Η Τ11II Π Ρ E Τ E11C AND AND 1. Pharmacologically active compounds 5'-deoxy-5-fluorocytidine or 5-deoxy-5-fluorouridine, as well as their physiologically acceptable acid addition salts. 2. 5'-Deoxy-5-fluorocyte hydidine according to claim 1. 3. 5'-deoxy-5-fluorouridine according to claim 1. A process for the preparation of a compound according to claim 1, more particularly of 5'-deoxy-5-fluorocytidine or 5'-deoxy5-fluorouridine, and their physiologically acceptable O acid addition salts, characterized in that the hydrolysis cleaves the 2 ', 3'-O-protecting group or cetalised to the 2', 3'-site of 5'-deoxy-5-fluorocytidine or 5'-deoxy- 5fluorouridine and optionally the resulting 5-deoxy-5-fluorocytidine or 5'-deoxy-5-fluorouridine is reacted with physiologically acceptable acid. The method of claim 4, wherein the 2 ', 3'-O-protected group is an alkylidene, cycloalkylidene or aralkylidene group which may optionally be substituted and in particular an isopropylidene group. The method according to claim 4, characterized in that 5'-deoxy-2 ', 3'-O-isoxypyrrolidine-5-fluorocytes and indine or 5' is 3 to 3 and - 2 ', 3 '- O - and z ο π ρ ο p i l i d e n - S - f l u ο ρ o o r p e d and n s e x i d ρ o l i z i p a with trifluoroacetic acid. 7. A pharmaceutical composition comprising a compound according to ironium 1, in particular S'deoxy-5-fluorocytiline or, respectively, N '- deoxy - N - fluorouridine, or a physiologically acceptable acid with one acid acid salt. one of these compounds and a non-toxic, inert, solid or liquid carrier suitable for therapeutic administration