CS270249B2 - Method of purine's new derivatives production - Google Patents

Description

The present invention relates to a process for the production of novel purine derivatives having valuable pharmacological properties.

It has been found that the novel purine derivatives of formula (I)

. i (I), in which

R ¹ represents a chlorine atom, an azido group or an amino group, and their amides and Schiff bases have useful pharmacodynamic properties. In particular, they have antiviral activity and can be used to treat or prevent viral diseases in humans and animals, particularly retroviral infections such as HIV and the like.

Accordingly, the present invention relates to the novel purine derivatives defined above as such and their use as therapeutically active agents. The invention further relates to a process for the manufacture of said compounds, of intermediates required for their manufacture, and of medicaments comprising said compounds, and of the use of defined compounds for the treatment or prevention of diseases, in particular for the treatment or prevention of viral diseases. .

Amides of the compounds of formula I are formed by substituting the amino group in position 6 and, if present, the substitution of amino compounds R ¹ listed in any convenient acyl group, like alkanoyl, e.g., acetyl group, propionyl group, butyryl group, pivaloyl group; an aroyl group such as a benzoyl group.

The Schiff bases of the compounds of formula I are similarly formed by treatment with any conventional aldehyde or ketone, such as benzaldehyde, or by treatment with dimethylformamide acetal.

Said compounds may exist in tautomeric forms. Such isomers are also within the scope of the present invention.

Particularly preferred compounds of formula I are the following:

6-amino-2-chloro-9- (2,3-dideoxy-2-fluoro- b -D-threopentofuranosyl) -9H-purine,

6-amino-2-azido-9- (2,3-dideoxy-2-fluoro-β-D-threopentofuranosyl) -9H-purine and 2,6-diamino-9- (2,3-diedoxy-2-fluoro) (D-D-threopentofuranosyl) -9H-purine.

The object of the present invention is a process for the preparation of the novel purine derivatives of the aforementioned general formula (Σ), comprising:

CS 270 249 B2 (II),

in which

R ¹ has the abovementioned meaning and

R represents a trityl group or a substituted trityl group, for example a monomethoxytrityl group, cleaves the R group, whereupon the optionally obtained compound of formula I is converted to an amide or a Schiff base.

The cleavage of the group R from the compound of the formula II can be carried out by methods known per se. For example, the cleavage can be carried out by treatment with an inorganic acid such as hydrochloric acid in a suitable solvent such as a halogenated aliphatic hydrocarbon such as chloroform, usually at temperatures of about 0 ° C to room temperature, or with a strong organic acid such as acetic acid, usually at an elevated temperature, for example at about 100 ° C.

The conversion of a compound of formula I into an amide or Schiff base can be carried out in a manner known per se, either by treatment with an appropriate acylating agent or by treatment with an appropriate aldehyde, ketone or acetal dimethylformamide.

The above-mentioned compounds of formula II which are used as starting materials in the process according to the invention can be prepared by first reacting a compound of formula III

(ni) in which

R ³ represents a C 1 -C 6 alkanoyl group, for example an acetyl group, or an aroyl group, for example a benzoyl group, and

R ⁴ is aroyl,

CS 270 249 B2 with ammonia, usually 8 ethanolic ammonia in a closed vessel at a temperature around room temperature for several days, for example about 7 days.

Then the 3 * -hydroxy group is removed from the resulting 6-amino-2-chloro-9- (2-deoxy-2-fluoro-D-arabinofuranosyl) -9H-purine of formula IV

Removal of the 3 * -hydroxy group from the compound of formula IV may be carried out in a manner known per se, first by converting the compound of formula IV to the compound of formula V in which:

R is as defined above.

(IN),

This conversion can be carried out in a known manner using trityl halide, for example trityl chloride, or substituted trityl halide in the presence of an acid binding agent, for example a tertiary amine, such as pyridine, usually at elevated temperature, for example at about 100 ° C.

The compound of formula (V) is then reacted with a phenylthionochloroformate, usually in an inert organic solvent such as acetonitrile, in the presence of an acid binding agent such as a tertiary amine such as pyridine, 4-dimethylaminopyridine, and at about room temperature. to form a compound of formula VI

CS 270 249 B2

(VI) in which:

R is as defined above and Ph is phenyl.

The compound of formula VI is then converted to a compound of formula II in which R ¹ is a chlorine atom by treatment with tributyltin hydride in the presence of a free radical initiator such as azoisobutyronitrile, usually in an inert organic solvent such as an aromatic hydrocarbon such benzene, toluene. , for example at a temperature of about 60 to 90 ° C.

The compound of formula (II) in which R1 is a chlorine atom can be converted to a compound of formula (II) in which it is an azido or amino group by methods known per se. For example, the chlorine atom R ^{1 may be} replaced by an azido group by reaction with hydrazine and subsequent reaction of the resulting hydrazino-substituted compound with an alkali metal nitrite such as sodium nitrite in the presence of a strong organic acid such as acetic acid. The R ¹ azido group can be converted to the amino group by catalytic hydrogenation in the presence of a palladium catalyst and an inert organic solvent, for example an alkanol such as ethanol, usually at about room temperature. Alternatively, the chlorine atom R1 can be replaced by an amino group by reaction with ammonia in an inert organic solvent such as a lower alkanol such as methanol at an elevated temperature, such as about 140 ° C, in a sealed tube.

The compounds of formula III above are known compounds or analogs of known compounds which can be prepared in an analogous manner to known compounds.

The compounds of formulas II, IV, V and VI mentioned above are novel compounds and are also an object of the present invention.

The efficacy of the purine derivatives obtained by the process of the invention against HIV can be demonstrated by the following experiment.

The experiment used HTLV-vir virus grown on C8166 cells (human CD ⁺ T lymphoblastoid line) using RPM1 1640 medium with bicarbonate buffer, antibiotics and 10% fetal bovine serum.

The cell suspension is infected ten times the virus a value and adsorption is performed for 90 minutes at 37 ° C. The cells are washed three times with medium. The test is performed in 6 ml tubes, each tube containing 2 x 10 6 infectoCS 270 249 B2 cells in 1.5 ml medium. Test compounds are dissolved either in aqueous medium or dimethylsulfoxide, depending on solubility, and 15 µL of test compound solution is added. The cultures are incubated at 37 ° C for 72 hours in a humidified atmosphere containing 5% carbon dioxide in air. The cultures are then centrifuged and an aliquot of the supernatant is dissolved with Nonidet P40 and subjected to antigen binding assay using a primary antiserum with particular reactivity to viral protein 24 and using horseradish peroxidase as a detection system. The resulting color is measured spectrophotometrically and plotted against test compound concentration. The concentration causing 50% protection (ΙΟ, ^ θ resp. IC ^ q) is determined.

To determine antiviral selectivity, a cytotoxicity assay based on dye consumption and metabolism or incorporation of a radiolabeled amino acid is performed in conjunction with the above assay.

The results obtained in the above experiment using selected compounds of formula I are summarized in the following table:

Table product from example no.

210

The purine derivatives of formula (I) produced by the process of the present invention can be used as medicaments in the form of pharmaceutical preparations containing them together with a compatible pharmaceutical carrier. *

These preparations may be administered orally, for example in the form of tablets, dragees, hard gelatine capsules, soft gelatine capsules, solutions, emulsions or suspensions; or parenterally, for example in the form of injectable solutions.

The above carrier material may be an inorganic or organic, pharmaceutically inert, carrier material. Examples of such carriers which may be used for the preparation of tablets, dragees and hard gelatin capsules include milk sugar, corn starch or derivatives thereof, talc, stearic acid or salts thereof. Examples of suitable carriers for soft gelatine capsules are vegetable oils, waxes, fats, semi-solid and liquid polyols. Suitable carriers for the preparation of solutions and syrups include, for example, water, polyols, sucrose, invert sugar and glucose. Suitable carriers for injectable solutions are, for example, water, alcohols, polyols, glycerol and vegetable oils. ‘

The pharmaceutical preparations may also contain conventional pharmaceutical auxiliaries such as preservatives, co-solvents, stabilizers, wetting agents, emulsifiers, sweeteners, coloring or flavoring agents, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. The pharmaceutical compositions may also contain other therapeutically valuable substances.

The dosage at which the novel purine derivatives produced according to the invention can be administered can vary within wide limits depending on the potency of the compound used, the condition being treated and the needs of the patient. This dose is determined by the attending physician. In general, the daily dose of the purine derivative may be about 0.1 to 20 mg / kg, preferably 0.2 to 15 mg / kg, and in particular about 0.4 to 10 mg / kg of body weight, although it will be appreciated that the stated range The doses are given by way of example only and can be varied in both the downward and upward directions.

CS 270 249 B2

The aforementioned purine derivatives may be administered in the form of a single dose or, in particular, in several sub-doses, for example up to six divided doses per day. A suitable dosage unit for this application may be, for example, from about 0.5 mg to 300 mg, preferably from about 1.0 mg to 300 mg, and in particular from about 2.0 mg to 200 mg of the purine derivative.

The pharmaceutical preparations can be prepared by mixing the above purine derivative and, if desired, one or more other therapeutically valuable substances with a compatible pharmaceutical carrier and, if desired, a pharmaceutical excipient and converting the mixture to the desired dosage form. The pharmaceutical preparations can be manufactured in a manner known per se.

The following examples illustrate the invention but do not limit it in any way.

Example 1

A solution of 0.39 g of 6-amino-2-chloro-9- (2,3-dideoxy-2-fluoro-5-O-trityl-β) -D-threopentofuranosylh9H-purine in 24 ml of absolute chloroform is cooled with ice and 2.2 ml of a 0.35M solution of hydrogen chloride in chloroform are then added thereto. The mixture was stirred at 0-5 ° C for 15 minutes, then at room temperature for 1.5 hours and then evaporated under reduced pressure. 20 ml of diethyl ether were added to the residue and the suspension was stirred for 30 minutes. The insoluble white solid was filtered off and washed with diethyl ether. 0.16 g of 6-amino-2-chloro-9- (2,3-dideoxy-2-fluoro-β-D-threopentofuranosyl) 9H-purine is obtained. The crude product obtained is dissolved in water and the solution is adsorbed on an ion exchange resin column (Zeocarb 225) in an H-cycle. After washing with water, the column was eluted with 2M ammonia solution. Evaporation of the eluate gave a white solid residue which was recrystallized from water to give analytically pure 6-amino-2-chloro-9- (2,3-dideoxy-2-fluoro-β-D-threopentofuranosyl) -9H-purine mp 210-211 ° C.

The 6-amino-2-chloro-9- (2,3-dideoxy-2-fluoro-5-O-trityl-β-D-threopentofuranosyl) -9H-purine used as starting material is prepared as follows:

A solution of 1.96 g of 2,6-dichloro-9- (3-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-O-D-arabionofuranosyl) -9H-purine in 195 ml of ethanol, was previously saturated at -5 ° C with ammonia, kept at room temperature in a closed stainless steel autoclave for 7 days. The solution is then evaporated under reduced pressure and the residue is recrystallized from 50 ml of ethanol. 0.38 g of 6-amino-2-chloro-9- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) -9H-purine is obtained, m.p. 212 DEG-215 DEG C. (decomposition). The mother liquor was evaporated under reduced pressure and the residue was purified by flash chromatography on silica gel eluting with 10% methanol in dichloromethane. A second crop (0.56 g) is obtained, m.p. 212 DEG-215 DEG C. (decomposition). A sample recrystallized from ethanol yields an analytically pure product with a melting point of 228 ° C.

A solution of 0.83 g of 6-amino-2-chloro-9- (2-deoxy-2-fluoro-4-D-arabinofuranosyl) -9H-purine in

7.5 ml of absolute pyridine was heated to 100 ° C under argon and a solution of 1.53 g of trityl chloride in 7.5 ml of absolute pyridine was added dropwise. The mixture was heated at 100 ° C for 2 hours and then evaporated under reduced ithac. The evaporation residue was partitioned between 100 ml of water and 100 ml of dichloromethane. The aqueous layer was separated and extracted twice with 50 ml of dichloromethane each. The combined organic phases were dried over sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using 5% methanol in dichloromethane as eluent. There was thus obtained 6-amino-2-chloro-9- (2-deoxy-2-fluoro-5-O-trityl-4-D-arabinofuranosyl) -9H-purine (0.84 g). Recrystallization from ethyl acetate gave an analytically pure product, m.p. 210-212 ° C.

Solution containing 0.73 g of 6-amino-2-chloro-9- (2-deoxy-2-fluoro-5-O-trityl-β-D-arabinofuranosyl) -9h-purine and 0.365 g of 4-dimethylaminopyridine in 21 ml of absolute acetonitrile was stirred at room temperature under argon and 0.20 ml of phenylCS 270 249-2 chlorothionocarbonate was added. The solution was stirred at room temperature overnight under argon. The solvent was removed by evaporation under reduced pressure and the residue was purified by flash chromatography on silica gel using 2% methanol in dichloromethane as eluent. 0.72 g of 6-amino-2-chloro-9- (2-deoxy-2-fluoro-3-O-phenoxythiocarbonyl-5-O-trityl-β-D-arabinofuranosyl) -9H-purine is obtained in the form of light yellow foam. ,

Solution containing 0,72 g of 6-amino-2-chloro-9- (2-deoxy-2-fluoro-3-O-phenoxythiocarbonyl-5-O-trityl-4-D-arabinofuranosyl) -9H-purine and 0, 04 g of azobisisobutyronitrile in 20 ml of absolute toluene was stirred under argon at room temperature and 0.42 ml of tributyltin hydride was added thereto. Argon was bubbled through the solution for 15 minutes and then heated to 75 ° C under argon for 2 hours. The toluene was removed by evaporation under reduced pressure and the residue was purified by flash chromatography on silica gel using 10% acetone in dichloromethane as eluent. There was obtained 0.45 g of 6-amino-2-chloro-9- (2,3-dideoxy-2-fluoro-5-O-trityl-1H-threopentofuranosyl) -9H-purine. Recrystallization from diethyl ether gave analytically pure product, m.p. 233-234 ° C.

Example 2.

mg of 6-amino-2-azido-9- (2,3-dideoxy-2-fluoro-5-O-trityl-1H-D-threopentofuranosyl) -9H-purine was heated at 100 ° C for 45 minutes in 5 ml of a mixture of 80% acetic acid and water. The mixture was evaporated and the residue was redissolved three times and evaporated using toluene. The residue was chromatographed on silica gel using dichloromethane / methanol 9: 1 as eluent. The fractions containing the desired product were collected and evaporated to give a gum which was dissolved in diethyl ether and the solution was evaporated again to give a solid. This solid was triturated twice with diethyl ether and the solid was collected by decantation. The residual solid was dried under reduced pressure to give 20 mg of 6-amino-2-azido-9- (2,3-dideoxy-2-fluoro). -D-threopentofuranosyl) -9H-purine, m.p. 202-204 ° C (decomposition) ·

The 6-amino-2-azido-9- (2,3-dideoxy-2-fluoro-5-O-trityl-4-D-threopentofuranosyl) -9H-purine used as starting material was prepared as follows:

132 mg of 6-amino-2-chloro-9F- (2,3-dideoxy-2-fluoro-5-O-trityl-1H-D-threopentofuranosyl) -9H-purine, prepared as described in Example 1, is added to 5 ml without aqueous hydrazine with stirring under a nitrogen atmosphere. The resulting suspension was stirred under a nitrogen atmosphere for 2 days. 10 ml of water were then added and the mixture was evaporated under reduced pressure. The residue was then evaporated again three more times using isopropyl alcohol in high vacuum. There was thus obtained 6-amino-2-hydrazino-9- (2,3-dideoxy-2-fluoro-5-O-trityl-4-D-threopentofuranosyl) -9H-purine as a white solid which was used without further purification. cleaning.

The above 6-amino-2-hydrazino-9- (2,3-dideoxy-2-fluoro-5-O-trityl-4-D-threopentofuranosyl) -9H-purine is dissolved in 15 ml of methanol and the solution obtained. 20 ml of water and then 2 ml of acetic acid are then added. The reaction mixture is stirred and then 21 mg of sodium nitrite is added while the solution is stirred. Stirring is continued until a solid precipitates. The mixture was allowed to stand at 4 ° C for 72 hours, then evaporated to a small volume and the residue partitioned between dichloromethane and water. The dichloromethane phase was evaporated to give a gum-like solid which still contained some of the above 2-hydrazino derivative. This gum-like solid was dissolved in a mixture of 20 ml of water, 30 ml of methanol, 20 ml of dichloromethane and 4 ml of acetic acid. 69 mg of sodium nitrite was added to the obtained solution. The solution is allowed to stand at room temperature for 30 minutes and then left to stand at 4 ° C for 2 hours. The solvents were removed by evaporation under reduced pressure and the residue was partitioned between dichloromethane and water. The dichloromethane phase is dried over anhydrous sodium sulphate and evaporated to give a solid which is purified by chromatography on silica gel using a mixture of dichloromethane and methanol.

CS 270 249 B2 9: 1. 100 mg of 6-amino-2-azido-9- (2,3-dideoxy-2-fluoro-5-O-trityl-4-D-threopentofuranosyl) -9H-purine is obtained as a light brown solid at a temperature of mp 220-222 ° C.

Example 3 mg of 2,6-diamino-9- (2,3-dideoxy-2-fluoro-5-O-trityl-β-D-threopentofuranosyl) -9H-purine was heated at 100 ° C for 1 hour with 10 ml of 80% acetic acid in water. The mixture was evaporated under reduced pressure and the residue redissolved twice and evaporated using toluene and twice using methanol. The residual solid was dissolved in a 9: 1 mixture of dichloromethane and methanol and this solution was chromatographed on silica gel. Eluting with a 9: 1 mixture of dichloromethane and methanol removes the by-product. The elution is then carried out with a 4: 1 mixture of dichloromethane and methanol to give fractions containing the desired product. These fractions were evaporated. The residue was redissolved and re-evaporated using diethyl ether. 30 mg of 2,6-diaroino-9- (2,3-dideoxy-2-fluoro-D-threopentofuranosyl) -9H-purine are obtained as a white solid, m.p. 235 DEG-237 DEG C. (decomposition). '

The 2,6-diamino-9- (2,3-dideoxy-2-fluoro-5-O-trityl- [beta] -D-threopentofuranosyl) -9H-purine used as starting material is prepared as follows:

A mixture of 0.131 g of 6-amino-2-chloro-9- (2,3-dideoxy-2-fluoro-5-O-trityl-1H-threopentofuranosyl) -9H-purine, prepared as described in Example 1, 7 ml of 33 % aqueous ammonia and 10 ml of methanol are stirred with a magnetic stirrer in a sealed tube at 140 ° C for 40 hours. The reaction mixture was evaporated and the resulting solid was redissolved and evaporated using ethanol and then using methanol. The residue was dissolved in a 9: 1 mixture of dichloromethane and methanol and purified by flash chromatography on silica gel using a 9: 1 mixture of dichloromethane and methanol as eluent. Fractions containing the desired product were combined and evaporated to give a cream colored solid. Dissolution in diethyl ether and evaporation gave 90 mg of 2,6-diamino-9- (2,3-dideoxy-2-fluoro-5-O-trityl-γ-D-threopentofuranosyl) -9H-purine as a white solid m.p. 226 DEG-230 DEG C. (decomposition), which is used without further purification.

The following examples illustrate typical pharmaceutical compositions which contain as an active ingredient purine derivatives which are prepared by the process of the present invention:

Example A

Tablets containing the following ingredients, active ingredient ingredients.

milk sugar starch. Polyvinylpyrrolidone magnesium stearate salt tablet weight can be prepared in the usual way per 1 mg mg mg mg

0.5 mg

0.5 mg mg '

CS 270 249 B2

Example of an active ingredient component »

milk sugar sodium starch glycolate magnesium stearic acid filling weight capsule per 1 mg mg

2.5 mg

0.5 mg mg

Claims (4)

SUBJECT OF THE INVENTION Process for the preparation of the novel purine derivatives of the general formula I (I), in which: R ¹ is chlorine, azido or amino, and their amides and Schiff bases, wherein »ee of compound of formula II (II)» wherein R ¹ has the abovementioned meaning and R is trityl or substituted trityl; CS 270 249 B2 cleaves the R group and the resulting compound of formula I is optionally converted into an amide or a Schiff base. 2. A process according to claim 1, wherein the corresponding compound of formula (II) is used as the starting material to give 6-amino-2-chloro-9- (2,3-dideoxy-2-fluoro- Β-D-threopentofuranosyl) -9H-purine. 3. The process of claim 1, wherein the corresponding compound of formula (II) is used as the starting material to provide 6-amino-2-azido-9- (2,3-dideoxy-2-fluoro-β-D-threopentofuranosyl). -9H-purine. 4. The process of claim 2, wherein the starting material is a corresponding compound of formula II to give 2,6-diamino-9- (2,3-dideoxy-2-fluoro-4-D-threopentofuranosyl) - 9H-purine.