NZ226672A - 6-amino-9-(2,3-dideoxy-2-fluoro-b-d-threopentofuranosyl)-9h-purine derivatives and pharmaceutical compositions - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £26672 22 6 6 7 2 NO DRAWINGS Priority cate(s):..

Complete Specification Filed: Class: 02-i ••••••••••••••••■•••••••••a WJUTWr Publication Date: P.O. Journal, No: NEW ZEALAND PATENTS ACT, 1953 No.: Date: COMPLETE SPECIFICATION PURINE DERIVATIVES .*! J* ■ J&; -t-/We. F. HOFFMANN-LA ROCHE & CO. AKTIENGESELLSCHAFT 124-184 Grenzacherstrasse, Basle, Switzerland, a Swiss company hereby declare the invention for which i-/ we pray that a patent may be granted to -na«/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - (followed by page la) 22 66 72 RAN 4430/28 The present invention relates to purine derivatives.

The purine derivatives provided by the present invention are compounds of the general formula wherein R1 represents chlorine, azido or amino, and amides and Schiff's bases thereof.

The purine derivatives aforesaid, i.e. the compounds of formula I and amides and Schiff's bases thereof, are 25 novel and possess valuable pharmacodynamic properties. In particular, they possess antiviral activity and can be used in the treatment or prophylaxis of viral infections of humans or animals, particularly of retroviral infections such as HIV and the like. 36 Me/31.8.88 Objects of the present invention are the purine derivatives defined above per se and for use as therapeutically active substances, a process for the manufacture of said derivatives, intermediates occurring in said process, medicaments containing said derivatives and 22 6 6 7 the use of said derivatives in the control or prevention of illnesses, especially in the treatment or prophylaxis of viral infections, or for the manufacture of medicaments for the treatment or prophylaxis of viral infections.

Amides of the compounds of formula I are formed by substitution of the 6-amino group and, where present, an amino group R1 of said compounds by any conventional acyl group such as an alkanoyl group (e.g. acetyl, propionyl, butyryl. pivaloyl) or an aroyl group (e.g. benzoyl). Schiff's bases of the compounds of formula I are likewise formed with any conventional aldehyde or ketone, such as benzaldehyde; or with a dimethylformamide acetal.

It will be appreciated that the compounds depicted herein can be present in tautomeric forms and that such tautomers are also included within the scope of the present invention.

Especially preferred compounds of formula I hereinbefore are: 6-Amino-2-chloro-9-(2,3-dideoxy-2-fluoro-6-D-threopento furanosyl)-9H-purine. 6-amino-2-azido-9-(2,3-dideoxy-2-fluoro-B-D-threopento-furanosyl)-9H-purine and 2,6-diamino-9-(2,3-dideoxy-2-fluoro-B-D-threopento-furanosyl)-9H-purine.

According to the process provided by the present invention, the purine derivatives aforesaid are manu- 2 factured by cleaving off the group R from a compound of the general formula 22 6 6 7 2 TjJ wherein R has the significance given earlier and 2 R represents trityl or substituted-trityl (e.g. monomethoxytr ityl). and. if desired, converting a compound of formula I obtained into an amide or into a Schiff's base. 2 The cleavage of the group R from a compound of formula II can be carried out according to methods known per se. For example, the cleavage can be carried out by treatment with an inorganic acid such as hydrogen chloride in a suitable solvent such as a halogenated aliphatic hydrocarbon (e.g. chloroform), conveniently at about 0°C to room temperature, or by treatment with a strong organic acid such as acetic acid, conveniently at an elevated temperature (e.g. about 100°C).

The conversion of a compound of formula I into an amide or into a Schiff's base can be carried out in a manner known per se by treatment with, respectively, an appropriate acylating agent or an appropriate aldehyde, ketone or dimethylformamide acetal. 22 6 6 7 2 The compounds of formula II above, which are used as starting materials in the process provided by the present invention, can be prepared by firstly treating a compound of the general formula CI 3 wherein R represents Cn-C.-alkanoyl (e.g. 4 acetyl) or aroyl (e.g. benzoyl) and R represents aroyl. with ammonia, conveniently with ethanolic ammonia in a sealed vessel at about room temperature for several days (e.g. about 7 days).

Subsequently, the 3'-hydroxy group is removed from the resulting 6-amino-2-chloro-9-(2-deoxy-2-fluoro-fl-D-ara-binofuranosyl)-9H-purine of the formula 22 6 6 7 2 nh2 iv. ho The removal of the 3'-hydroxy group from the compound of formula IV can be carried out in a manner known per se by firstly converting the compound of formula IV into a compound of the general formula This conversion can be carried out in a known manner using a trityl halide (e.g. trityl chloride) or a substituted-trityl halide in the presence of an acid--binding agent (e.g. a tertiary amine such as pyridine), conveniently at an elevated temperature (e.g. about 100°C). nh2 V ho 2 wherein R has the significance given earlier. 22 6 6 7 2 A compound of formula V is subsequently reacted with phenyl thionochloroformate, conveniently in an inert organic solvent such as acetonitrile. in the presence of an acid-binding agent such as a tertiary amine (e.g. pyridine. 4-dimethylaminopyridine) and at about room temperature to give a compound of the general formula VI 2 wherein R has the significance given earlier and Ph represents phenyl.

A compound of formula VI is then converted into a compound of formula II in which R1 represents chlorine by treatment with tributyltin hydride in the presence of a free radical initiator (e.g. azoisobutyronitrile), conveniently in an inert organic solvent such as an aromatic hydrocarbon (e.g. benzene, toluene) and at an elevated temperature (e.g. about 60°-90°C).

A compound of formula II in which R1 represents chlorine can be converted into a compound of formula II in which R1 represents azido or amino in accordance with methods known per se. Thus, for example, the chlorine atom R1 can be replaced by azido by reaction with hydrazine and subsequent treatment of the resulting hydrazino-sub- 22 6 6 7 r stituted compound with an alkali metal nitrite (e.g. sodium nitrite) in the present of a strong organic acid such as acetic acid. An azido group R1 can be transformed into an amino group by catalytic hydrogenation in the presence of a palladium catalyst and in an inert organic solvent (e.g. an alkanol such as ethanol). conveniently 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 (e.g. methanol) at an elevated temperature (e.g. about 140°C) in a sealed tube.

The compounds of formula III hereinbefore are known compounds or analogues of known compounds which can be prepared in an analogous manner to the known compounds.

The compounds of formulae II, IV, V and VI hereinbefore are novel and also form objects of the present invention.

The activity of the purine derivatives provided by the present invention against HIV can be demonstrated in the assay described hereinafter.

This assay uses HTLV-III grown in C8166 cells (a human CD4+ T lymphoblastoid line) using RPM1 1640 medium with bicarbonate buffer, antibiotics and 10% foetal bovine serum.

A suspension of cells is infected with ten times the TCDc_ of virus and adsorption allowed to proceed for DU 90 minutes at 37°C. The cells are washed three times with medium. The test is carried out in 6 ml tissue culture tubes, each tube containing 2 x 10 infected cells in 1.5 ml of medium. Test substances are dissolved in either aqueous medium or dimethyl sulphoxide, according to solubility, and a 15 y.1 solution of the substance added. 226672 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 solubilized with Nonidet P40 and subjected to an antigen capture assay which uses a primary antiserum with particular reactivity against the viral protein 24 and a horseradish peroxidase detection system. Colour generation is measured spectrophotometrically and plotted against the concentration of test substance. The concentration that produces 50% protection is determined (IC5Q and IC9Q respectively).

A cytotoxicity assay based on dye uptake and metabolism or radio-labelled amino acid incorporation is run alongside the above assay in order to determine antiviral selectivity.

The results obtained in the foregoing assay using representative compounds of formula I are compiled in the following Table: Table Product of Example No.

IC5Q (vM) 1 1 2 3 1 22 6 6 72 The purine derivatives provided by the present invention can be used as medicaments in the form of pharmaceutical preparations which contain them in association with a compatible pharmaceutical carrier material.

The pharmaceutical preparations can be administered orally (e.g. in the form of tablets, dragees, hard gelatine capsules, soft gelatine capsules, solutions, emulsions or suspensions) or parenterally (e.g. in the form of injection solutions).

The carrier material present in the pharmaceutical preparations can be a pharmaceutically inert, inorganic or organic carrier. Examples of such carriers which can be used for tablets, dragees and hard gelatine capsules are lactose, maize starch or derivatives thereof, talc, stearic acid or its salts. Examples of suitable carriers for soft gelatine capsules are vegetable oils, waxes. fats, semi-solid and liquid polyols etc. Suitable carriers for the production of solutions and syrups include, for example, water, polyols. saccharose, invert sugar, glucose and the like. Suitable carriers for injection solutions are, for example, water, alcohols, polyols, glycerine, vegetable oils.

The pharmaceutical preparations can also contain conventional pharmaceutical adjuvants such as preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifying agents, sweetening agents, colouring agents, flavouring agents, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. The present pharmaceutical preparations can contain other therapeutically valuable substances.

The dosage in which the present purine derivatives can be administered will vary according to the potency of the 22 6 6 7 2 particular derivative in question, the condition being treated and the requirements of the patient as determined by the attending physician. In general, the purine derivatives can be administered in a daily dosage of about 0.1 mg to about 20 mg/kg body weight, preferably about 0.2 mg to about 15 mg/kg body weight and particularly about 0.4 mg to about 10 mg/kg body weight, although it will be appreciated that these dosage ranges are given by way of example only and may be varied upwards or downwards.

The purine derivatives provided by the present invention can be administered in a single dosage or. preferably, in several sub-dosages (e.g. up to six) divided over the day. A suitable unit dosage form for this administration can contain, for example, from about 0.5 mg to about 300 mg, preferably about 1.0 mg to about 300 mg and particularly about 2.0 mg to about 200 mg of a purine derivative of this invention.

The pharmaceutical preparations can be produced by mixing a purine derivative aforesaid and, if desired, one or more other therapeutically valuable substances with a compatible pharmaceutical carrier material and, if desired, a pharmaceutical adjuvant and bringing the mixture into a desired administration form. The production of the pharmaceutical preparations can be carried out in a manner known per se.

The following Example illustrates the present invention in more detail: Example 1 A solution containing 0.39 g of 6-amino-2-chloro-9--(2.3-dideoxy-2-fluoro -5-O-trityl-B-D-threopento-furanosyl)-9H-purine in 24 ml of dry chloroform was cooled in ice and treated with 2.2 ml of a 0.35M solution of 22 6 6 7 2 hydrogen chloride in chloroform. The mixture was stirred at 0-5°C for 15 minutes, then at room temperature for 1.5 hours and subsequently evaporated under reduced pressure. The residue was treated with 20 ml of diethyl ether and the suspension was stirred for 30 minutes. The insoluble white solid was filtered off and washed with diethyl ether to give 0.16 g of 6-amino-2-chloro-9-(2,3--dideoxy-2-fluoro-fl-D-threopentofuranosyl)-9H-purine- This crude product was dissolved in water and absorbed on to a column of Zeocarb 225 cation exchange resin (H+ form). 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-B-D-threopentofuranosyl)-9H-purine of melting point 210-211°C.

The 6-amino-2-chloro-9-(2.3-dideoxy-2-fluoro-5-O--trityl-B-D-threopentofuranosyl)-9H-purine used as the starting material was prepared as follows: A solution of 1.96 g of 2,6-dichloro-9-(3-0-acetyl-5--0-benzoyl-2 -deoxy-2-fluoro-B-D-arabinofuranosyl)-9H--purine in 195 ml of ethanol, which has previously been saturated with ammonia at -5°C, was held at room temperature for 7 days in a sealed stainless steel autoclave. The solution was evaporated under reduced pressure and the residue was recrystallized from 50 ml of ethanol to give 0.38 g of 6-amino-2-chloro-9-(2-deoxy-2-fluoro-B -D-ara-binofuranosyl)-9H-purine of melting point 212-215°C (decomposition). The mother liquors were evaporated under reduced pressure and the residue was purified by flash chromatography on silica gel using 10% methanol in di-chloromethane for the elution. There was thus obtained a second portion of 0.56 g of the desired product of melting point 212-215°C (decomposition). A sample was recrystallized from ethanol to give analytically pure material of 22 6 6 7 2 r melting point 228°C.

A solution of 0.83 g of 6-amino-2-chloro-9-(2-deoxy-2--fluoro-B-D-arabinofuranosyl)-9H-purine in 7.5 ml of dry pyridine was heated at 100°C under argon while a solution of 1.53 g of trityl chloride in 7.5 ml of dry pyridine was added dropwise. The mixture was heated at 100°C for 2 hours and then evaporated under reduced pressure. The 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 time. The combined organic phases were dried over sodium sulphate, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using 5% methanol in dichloromethane for the elution. There was obtained 0.84 g of 6-amino-2-chloro-9-(2-deoxy--2-fluoro-5-0-trityl -fl-D-arabinofuranosyl)-9H-purine. Recrystallization from ethyl acetate gave analytically pure product of melting point 210-212°C.

A solution containing 0.73 g of 6-amino-2-chloro-9-(2--deoxy-2-fluoro-5 -O-trityl-fl-D-arabinofuranosyl)-9H--purine and 0.365 g of 4-dimethylaminopyridine in 21 ml of dry acetonitrile was stirred at room temperature under argon and treated with 0.20 ml of phenyl chlorothiono-carbonate. The solution was stirred at room temperature under argon overnight. 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 for the elution. There was obtained 0.72 g of 6-amino-2-chloro-9-(2-deoxy-2-fluoro-3 -O-phenoxythio-carbonyl-5-0-trityl-B-D-arabinofuranosyl)-9H-purine as a pale yellow foam.

A solution containing 0.72 g of 6-amino-2-chloro-9-(2--deoxy-2-fluoro-3 -O-phenoxythiocarbonyl-5-O-trityl-fl-D--arabinofuranosyl)-9H-purine and 0.04 g of azobisiso- 22 6 6 7 butyronitrile in 20 ml of dry toluene was stirred under argon at room temperature and treated with 0.42 ml of tributyltin hydride. Argon was bubbled through the solution for 15 minutes and the solution was then heated at 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 for the elution. There was obtained 0.45 g of 6-amino-2-chloro-9-(2,3-dideoxy-2--fluoro-5 -O-trityl-B-D-threopentofuranosyl)-9H-purine. Recrystallization from diethyl ether gave analytically pure product of melting point 233-234°C.

Example 2 75 mg of 6-amino-2-azido-9-(2,3-dideoxy-2 -fluoro-5-O--trityl-fl-D-threopentofuranosyl)-9H-purine were heated at 100°C for 45 minutes in 5 ml of 80% acetic acid/water. The mixture was evaporated and the residue was re-evaporated three times with toluene. The residue was chromatographed on silica gel using dichloromethane/methanol (9:1) for the elution. The product-containing fractions were combined and evaporated to give a gum which was re-evaporated with diethyl ether to give a solid. This solid was triturated twice with diethyl ether, the solvent being removed by decantation. The residual solid was dried in a vacuum to give 20 mg of 6-amino-2-azido-9-(2,3-dideoxy-2-fluoro-B-D--threopentofuranosyl)-9H-purine of melting point 202-204°C (decomposition).

The 6-amino-2-azido-9-(2.3-dideoxy-2 -fluoro-5-O--trityl-B-D-threopentofuranosyl)-9H-purine used as the starting material was prepared as follows: 132 mg of 6-amino-2-chloro-9-(2.3-dideoxy-2-fluoro-5--0-trityl-B-D-threopentofuranosyl)-9H-purine. prepared as described in Example 1 were added to 5 ml of anhydrous 22 6 6 7 hydrazine stirred under nitrogen. The resulting slurry was stirred for 2 days under nitrogen. 10 ml of water were then added and the mixture was evaporated under a vacuum into a receiver containing water. The residue was re--evaporated three times with isopropanol under a high vacuum to give 6-amino-2-hydrazino-9-(2,3-dideoxy-2--fluoro-5-O-trityl-B-D-threopentofuranosyl)-9H-purine as a white solid which was used without further purification.

The foregoing 6-amino-2-hydrazino-9-(2,3-dideoxy-2--fluoro-5-0-trityl-B-D-threopentofuranosyl)-9H-purine was dissolved in 15 ml of methanol and there were then added 20 ml of water followed by 2 ml of acetic acid. The mixture was stirred and 21 mg of sodium nitrite were added to give a solution. As stirring was continued some solid was precipitated. The mixture was left to stand at 4°C for 72 hours, then evaporated to a small volume and partitioned between dichloromethane and water. The dichloromethane phase was evaporated to give a gum-like solid which still contained some aforementioned 2-hydra-zino compound. 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 were then added, whereby a solution was obtained. This solution was left to stand at room temperature for 30 minutes and then at 4°C for 2 hours. The solvents were removed by evaporation in a vacuum and the residue was partitioned between dichloromethane and water. The dichloromethane phase was dried over anhydrous sodium sulphate and evaporated to give a solid which was purified by chromatography on silica gel using dichloromethane/ methanol (9:1) for the elution. There were obtained 100 mg of 6-amino-2-azido-9-(2,3-dideoxy-2 -fluoro-5-O-trityl-B--D-threopentofuranosyl)-9H-purine as a fawn coloured solid of melting point 220-222°C. 22 6 6 7 2 Example 3 90 mg of 2.6-diamino-9-(2,3-dideoxy-2-fluoro-5-O--trityl-B-D-threopentofuranosyl)-9H-purine were heated at 100°C for 1 hour with 10 ml of 80% acetic acid/water. The mixture was evaporated in a vacuum and the residue was re-evaporated twice with toluene and twice with methanol. The residual solid was dissolved in dichloromethane/ methanol (9:1) and chromatographed on silica gel. Elution with dichloromethane/methanol (9:1) removed a byproduct. Subsequent elution with dichloromethane/methanol (4:1) yielded fractions which contained the desired product and which were evaporated. The residue was re-evaporated with diethyl ether to give 30 mg of 2.6-diamino-9-(2,3-dideoxy--2-fluoro-B-D-threopentofuranosyl)-9H-purine as a white solid of melting point 235-237°C (decomposition).

The 2.6-diamino-9-(2.3-dideoxy-2-fluoro-5-O-trityl-B--D-threopentofuranosyl)-9H-purine used as the starting material was prepared as follows: A mixture of 0.131 g of 6-amino-2-chloro-9-(2.3--dideoxy-2-fluoro-5-0-trityl-B -D-threopentofuranosyl)-9H--purine. prepared as described in Example 1. 7 ml of 33% aqueous ammonia and 10 ml of methanol was stirred magnetically in a sealed Carius tube at 140°C for 40 hours. The mixture was evaporated and the resulting solid was re--evaporated with ethanol and then with methanol. The residue was dissolved in dichloromethane/methanol (9:1) and purified by flash chromatography on silica gel using dichloromethane/methanol (9:1) for the elution. The product-containing fractions were combined and evaporated to give a cream coloured solid. Re-evaporation with diethyl ether gave 90 mg of 2,6-diamino-9-(2,3-dideoxy-2--fluoro-5-0 -trityl-B-D-threopentofuranosyl)-9H-purine as a white solid of melting point 226-230°C (decomposition)

Claims (17)

- 16 - 22 6 6 7 which was used without further purification. The following Examples illustrate typical pharmaceutical preparations containing the purine derivatives provided by the invention as the active ingredient: Example A Tablets containing the following ingredients may be prepared in a conventional manner: Ingredients Per tablet Active ingredient Lactose Starch Polyvinylpyrrolidone Magnesium stearate Tablet weight 10 mg 20 mg 4 mg 0.5 mg 0.5 mg 35 mg Example B Ingredients Per capsule Active ingredient Lactose Sodium starch glycollate Magnesium stearate 25 mg 15 mg 2.5 mg 0.5 mg Capsule fill weight 43 mg whattfwe claim is- CLAIMS \ - 17 - 22 6 6 7 2

1. Compounds of the general formula 5 "4 I I 10 R HO 15 wherein R1 represents chlorine, azido or amino and amides and Schiff's bases thereof.

2. 6-Amino-2-chloro-9-(2.3-dideoxy-2-fluoro-B-D-threopento-furanosyl)-9H-pur ine. 20

3. 6-Amino-2-azido-9-(2,3-dideoxy-2-fluoro-B-D-threopentofuranosyl )-9H-pur ine.

4. 2,6-Diamino-9-(2,3-dideoxy-2-fluoro-B-D-threopento-25 furanosyl)-9H-purine. 30 35 21 6 6 7 2 - 18 -

5. Compounds of the general formula ~Xj wherein R represents chlorine, azido or amino and 2 R represents trityl or substituted-trityl.

6. 6-Amino-2-chloro-9-(2-deoxy-2-fluoro-B-D-arabinofurano-syl)-9H-purine.

7. Compounds of the general formula tJ ho 2 wherein R represents trityl or substituted-trityl. 22 6 6 7 2 m O G - 19 -

8. Compounds of the general formula 10 N VI s=cCh 15 2 wherein R represents trityl or substituted-trityl and Ph represents phenyl.

9. A purine derivative according to any one of claims 1 20 to 4 for use as a therapeutically active substance.

10. A purine derivative according to any one of claims 1 to 4 for use in the treatment or prophylaxis of viral /"""N infections, especially of retroviral infections and 25 particularly of HIV infections.

11. A process for the manufacture of the purine derivatives as claimed in any one of claims 1 to 4, which 2 process comprises cleaving off the group R from a 30 compound of the general formula 35 22 6 672 - 20 - wherein R1 has the significance given earlier and 2 . . R represents trityl or substituted-trityl. and, if desired, converting a compound of formula I obtained into an amide or into a Schiff's base.

12. A medicament containing a purine derivative set forth in any one of claims 1 to 4 and a compatible pharmaceutical carrier material.

13. A medicament for the treatment or prophylaxis of viral infections, especially of retroviral infections and particularly of HIV infections, containing a purine derivative set forth in any one of claims 1 to 4 and a compatible pharmaceutical carrier material. - 21 - Mum I

14. The use of a purine derivative set forth in any one of claims 1 to 4 for the manufacture of a medicament for the treatment or prophylaxis of viral infections, especially of retroviral infections and particularly of HIV infections.

15. A process for the manufacture of the purine derivatives as claimed in any one of claims 1 to 4, substantially as hereinbefore described with reference to any one of Examples 1 to 3.

16. A medicament containing a purine derivative set forth in any one of claims 1 to 4 and a compatible pharmaceutical carrier material, substantially as hereinbefore described with reference to either of Examples A and B.

17. A purine derivative according to any one of claims 1 to 4 whenever prepared by a process according to claim 11 or claim 15. 24 MAY199IS) / 19