NO782648L - CYCLIC CONNECTION. - Google Patents

Description

Cyclic connection.

The present invention relates to a hitherto unknown cyclic compound, namely 1-methyl-9(3D-ribofuranosyl-isoguanine (1-methyl-isoguanosine) which can exist in tautomeric forms, e.g. with the formula

According to the invention, it was found that 1-methyl-isoguanosine is present in small quantities in organisms in the sea, especially in sponges of the genus Tedania found on the Australian coast.

In one respect, the invention relates to the isolation of 1-methyl-isoguanosine from natural occurrences, in particular organisms in the sea, and in particular sponges of the genus Tedania.

Furthermore, the invention relates to 1-methyl-isoguanosine even in practically pure form, i.e. free from naturally occurring contaminants.

1-methyl-isoguanosine can be isolated from natural occurrences, e.g. sponges, in a manner known per se, e.g. by extraction. with dimethylsulfoxide, water or an organic hydroxylated solvent such as a lower alkanol, e.g. methanol or ethanol under which an ethanol-water mixture is preferred and subsequent chromatography, preferably ion exchange chromatography, e.g. by means of a cation-exchange resin such as an SO^H group-containing resin and a weakly acidic buffer such as ammonium formate.

The invention further relates to a method for the production of 1-methyl-isoguanosine by reacting an imidazole derivative with the formula

where R is an easily cleavable acyl group,

with methyl isocyanate. in the presence of a strong aprotic solvent such as dimethylformamide, dimethylsulfoxide or hexamethylphosphoramide, preferably at a temperature between room temperature and 150°C, preferably at approx. 100°C.

This reaction produced a new intermediate product with formula

where R has the above meaning,

is then reacted with a base, preferably a weak base such as ammonium hydroxide in the presence or without a C^_^-alkanol, with anhydrous ammonia in the presence of a C-^^-alkanol, or with an alkali metal hydroxide, e.g. sodium or potassium hydroxide. This reaction is preferably carried out at a temperature between 0 and 100°C, preferably at room temperature.

Examples of easily cleavable acyl groups R in the starting materials of formula II are lower alkanoyl such as acetyl, propionyl or butyryl, aroyl or substituted aroyl such as benzoyl, p-tolyoyl, p-methoxybenzoyl or p-chlorobenzoyl, preferably acetyl. j

Furthermore, the invention relates to a method for the production of 1-methyl-isoguanosine by reacting a compound with formula

with a methylating agent.

The methylation is preferably carried out using a methyl halide, e.g. methyl iodide or bromide, in the presence of an aprotic solvent such as dimethylformamide, dimethylsulfoxide or hexamethylphosphoramide under basic conditions, e.g.

in the presence of sodium or potassium hydroxide or carbonate, at a temperature between approx. 0 and 100°C, preferably around room temperature. As a by-product of the above-mentioned methylation, it is obtained from J.Org. Chem. 22, 1957, 1575 known 9p>-D-ribofuranosyl-2-methoxy-adenine (2-methoxy-adenosine.) . The products can be separated in a manner known per se, e.g. by chromatography.

The methylation in the 1-position of the isoguanosine IV can also be carried out using diazomethane in a solvent such as an ether, e.g. dioxane or 1,2-dimethoxyethane, or a C^_^-alkanol, e.g. ethanol. As a methylating agent, dimethyl sulfate in a polar aprotic solvent can also be used as dioxane or in water. These reactions are preferably carried out at around room temperature.

According to the invention, it was further found that 1-methyl-isoguanosine has various therapeutic effects, e.g. has a muscle-relaxing effect, affects the central nervous system, is anti-inflammatory, anti-allergic and hypotensive. In mice, the EDj-Q value for 1-methyl-1-isoguanosine as a muscle relaxant by intraperitoneal application is 3.1 mg per kg and by oral application 12 mg pir. kg. LD5q ^os mouse is 10 00 mg per kg p.o. after 4 8 hours.

1-methyl-isoguanosine can be used as a muscle relaxant and/or as an agent1 for influencing the central nervous system and/or as an anti-inflammatory and/or anti-allergic and/or hypotensive agent. It can be used in the form of pharmaceutical preparations together with a tolerable pharmaceutical carrier, e.g. one organic or inorganic inert carrier suitable for enteral, preferably oral or parenteral administration. Examples of such carriers are water, gelatin, . lactose, starch, talc, magnesium stearate, gum, vegetable oils and petroleum jelly.

The pharmaceutical preparations can be in solid form, e.g. as tablets, capsules, dragees or suppositories or in liquid form, e.g. as solutions, emulsions or suspensions. They may be sterilized and/or contain tolerable aids such as preservatives, stabilisers, flavourings, colourants, emulsifiers and salts for changing the osmotic pressure.

A suitable pharmaceutical dosage form contains approx. 1-100 mg of 1-methyl-isoguanosine. The dosage for oral administration is in the range of 0.1 mg per kg to 25 mg per kg per day. For parenteral administration, dosages in the range from 0.01 mg per kg to 10 mg per kg per day. However, these areas can be varied upwards or downwards depending on the individual cases.

The following examples illustrate the invention.

Example 1

The starting material was an orange sponge of the genus Tedania that was collected on the Australian coast. The deep-frozen organism was lyophilized and ground. The. the powder obtained (300 g) was stirred with an ethanol:water mixture (3:7 v/v, 2 L) for 24 h at 4°C, then filtered and the residue re-extracted. The combined extracts were concentrated at 35°C and 7 torr

a volume of 1.2 1 and the thus obtained aqueous suspension centrifuged for 0.3 hours at 13,000 g and the supernatant lyophilized. 75.7 g of orange colored powder A was thereby obtained.

205 g of the crude extract A was suspended in 0.1 M ammonium formate buffer (pH 3.5), 15 min. ultrasonically irradiated■and 40 min. stirred in a boiling water bath. The suspension was cooled to 22°C, pH brought to 4.2. to 3.5 by adding formic acid and then centrifuged for half an hour at 13000 g. The supernatant was applied to a column (40 x 5 cm) of Bio Rad AG50W-X8 ammonium form, 200-400 Mesh) and equilibrated with 0.1 M ammonium formate (pH 3.5) with a flow rate of 150 ml per hour. Below that, the percentage permeability of the eluate at 254 bg 280 nm was monitored. After 10 1 of buffer had run through, 0.1 M ammonium formate (pH 5.3) was placed on the column. The following fractions were collected: 1.2 1 (pH 3.5-4.8) 4.5.1 (pH 4.8-5.0) and 5 1 (pH 5.0-5.2). Only the fraction with pH 4.8-5.0 was active, it was lyophilized and gave a pale yellow solid B (31.6 g).

A solution of 68.9 g B in 1.25 1 0.1 M ammonium formate (pH 3.5) was centrifuged for 20 min. at 13,000 g. The supernatant was diluted with 0.1 M ammonium formate (pH 3.5) to 4 1 and the pH was adjusted to 3.7 by adding formic acid. The solution was applied to a column (41.5 x 5 cm) of Bio Rad AG50W-X8 (ammonium form, 200-400 Mesh) and the above procedure for the treatment of crude extract A was repeated. After that 5.1 1 pH

3.5 buffer had been eluted, buffer with pH 5.3 was put on 1 column and the active material eluted between 7.85 1 and 10.85 1. Lyophilization of the active fraction gave 10.8 g of C.

Recrystallization of C from boiling water gave 2.75 g of 1-methylisoguanosine as a colorless crystalline solid, mp 262-263°C, [a] ^gg - 65.4° (c = 1.0, dimethylsulfoxide) [ algjjg - 54.6°

(c = 1.0, water).

Example 2

1- methyl isoguanosine

A solution of 5-amino-4-cyano-1-(2<1>,3',51-tri-O-acetyl-ft-D-ribofuranosyl)imidazole (25.4 g, 69.3 mmol) and methyl isocyanate (40.9 ml, 693 mmol) in DMF (250 ml, dried over 4A molecular sieve> heated with stirring at 100°C. After 6 hours the mixture is cooled and evaporated to an oil. Traces of DMF are removed by evaporation together with 250 ml of methanol. The foam formed is dissolved in 250 ml of methanol, filtered and evaporated to dryness. The foam is left standing under vacuum, then dissolved in 150 ml of methanol and treated overnight at 5°C with 150 ml of concentrated ammonium hydroxide. The formed the crystals are collected, washed with methanol and recrystallized from ethanol/water (1/1). Traces of impurities are removed by repeated recrystallizations. Melting point 265-266°C cleavage.

Example 3

A solution of 5 g of 5-amino-4-cyano-1-(2',3',5'-tri-O-acetyl-|3-D-ribofuranosyl)imidazole in 50 ml of DMF dried over a 4A molecular sieve is treated with stirring for 23 hours under reflux at 100°C with 8.05 ml of methyl isocyanate. The solution is evaporated to dryness, evaporated together with 2 x 50 ml of methanol and allowed to stand overnight under vacuum. 4.76 g of the foam obtained is dissolved in 48 ml of ethyl acetate/chloroform (3/1) and chromatographed over silica gel with acetate/chloroform (3/1) as eluent. Fractions of 20 ml are collected. Fractions 122-160 are combined and evaporated to a white foam. For purification, this foam is dissolved in 10 ml of chloroform/methanol (50/1) and chromatographed over silica gel with chloroform/methanol (50/1) as eluent. 20 ml<1>s the fractions 100-140' are evaporated to dryness and 4-cyano-5-i[bis-(methyl-carbamoyl)]-amino-1-(2 ' , 3 1 , 5 ' -tri- O-acetyl-pj-D- ■

ribofuranosyl)imidazole as white foam; IR 2245 cm (C=sN), 1753 (Ester), 1728, 1680 (ureacarbonyl); UV 227 nm (e11,360)

in methanol, 226 nm (£9540) in 0.1N HCl.

A solution of 480 ml of 4-cyano-5-[bis-(methylcarbamoyl)]-amino-1-(2'; 3', 5-tri-O-acetyl-β-D-ribofuranosyl)-imidazole in .5 ml of methanol and 5 ml of concentrated ammonium hydroxide is stored for 18 hours at 5°C. The product is evaporated to dryness and recrystallized from ethanol/water. This gives 1-methylisoguanosine with a melting point of 267-268°C (decomposition).

Example 4

A solution of 200 mg of isoguanosine in 6 ml of dimethylsulfoxide dried over a 4A molecular sieve is stirred for 18 hours at room temperature with 215 mg of potassium carbonate and 0.088 ml of methyl iodide. The mixture is filtered, evaporated to an oil, the latter is dissolved in methanol/water (9/1) and placed on 7 g of silicon oxide. The silicon oxide is dried by evaporation of 3 x 100 ml of ethanol, allowed to stand overnight in a vacuum and placed on a silicon oxy column. The column is eluted with 750 ml of chloroform/methanol (5/1) and then with chloroform/methanol (3/1). 20 ml fractions 45-60 are collected and evaporated. You get raw 2- • methoxy-adenosine. After recrystallization from water, a pure product with a melting point of 189-192° is obtained.

Fractions 100-140 are evaporated to dryness. You get crude 1-methyl-isoguanosine. Part of this is purified by preparative thin-layer chromatography on silicon oxide with chloroform/methanol (2/1). The suitable portion is extracted with water. The extract is evaporated to dryness and dissolved in hot water. Small amounts of silicon oxide are removed by filtration. The filtrate is evaporated to 0.1 ml and mixed with 0.2 ml of ethanol. The mixture is kept overnight at 5°C and crystals with a melting point of 265°C (decomposition) are obtained.

Example 5

Tablets with the following composition are produced in the usual way:

Claims (14)

1. Process for the production of 1-methylisoguanosine, characterized in that one a) isolates it from natural occurrences in a manner known per se or b) reacts a compound with the formula where R is an easily cleavable acyl group with methyl isocyanate in the presence of a strong aprotic solvent and reacts the obtained compound with the formula where R has the above meaning, with a base or c) reacts a compound of formula with a methylating agent. 2. Method according to claim 1, characterized in that method variant a) is carried out. 3. Method according to claim 1, characterized in that method variant b) is carried out. 4. Method according to claim 1, characterized in that method variant c) is carried out. 5. Method' according to claim 1 or 2, characterized in that a marine organism is used as a naturally occurring material and 1-methyl-isoguanosine is isolated by extraction with a solvent and subsequent chromatography. 6. Method according to claim 5, characterized in that a mixture of water and ethanol is used as solvent and the extract is purified by ion exchange chromatography. 7. Process according to claim 1 or 3, characterized in that Acetyl is used as the acyl group R. 8. Method according to claim 1 or 4, characterized in that the methylation of the compound of formula IV. is carried out with methyl halide in the presence of an aprotic solvent under basic conditions. 9. Process for the production of pharmaceutical preparations, characterized by mixing 1-methyl-isoguanosine as an active ingredient with non-toxic inert suitable for therapeutic administration in and of such preparations usual solid and liquid carriers and/or excipients. 10. Pharmaceutical preparations, characterized by a content of 1-methyl-isoguanosine. 11. Compounds with the formula characterized in that R is an easily cleavable acyl group. 12. Compound according to claim 11, characterized in that R is acetyl. 13. 1-Methylisoguanosine. 14. 1-methyl-isoguanosine in practically pure form free of naturally occurring impurities.