DK165985B - 1-CYTOSIN DERIVATIVES, PROCEDURES FOR THEIR PREPARATION, AND THE USE OF THEM IN AND FOR THE PREPARATION OF MEDICINAL PRODUCTS - Google Patents

Description

in

DK 165985B

The present invention relates to 1-cytosine derivatives of the general formula I

NH,

A

5 wherein R 1 represents hydroxy, alkanoyloxy or aroyloxy, and amides of the compounds of formula I formed by the 4-amino group having an alkanoyl or aroyl group.

Surprisingly, these compounds have been found to have antiviral activity and can be used for the treatment or prophylaxis of viral infections, especially retroviral infections such as sheep lentiviruses or HIV infections and the like.

In particular, the present invention relates to the above-defined compounds for use as therapeutic active substances; a process for preparing these compounds; drugs containing these compounds, and the use of these compounds in the manufacture of drugs for the treatment or prophylaxis of viral infections, especially retroviral infections, especially HIV infections.

Examples of alkanoyloxy groups R 1 are acetoxy, propionyloxy or butyryloxy; Examples of aroyloxy groups are benzoyl-oxy or substituted benzoyloxy, for example p-toluoyloxy or p-chlorobenzoyloxy. Amides of the compounds of formula I are formed at the 4-amino group with any conventional alkanoyl group, for example acetyl, propionyl, butyryl or pivaloyl; or aroyl group, e.g. benzoyl.

DK 165985B

2- 1- (2 ', 3' -Dideoxy-2 '-fluoro- / S -D-arabinofuranosy 1) cytosine (hereinafter referred to as DFAC) is a particularly preferred compound of formula I,

The process according to the invention is characterized in that the 5 (a) 3 'hydroxy group in a compound with the general

formula II

NH2 /) in 11

HAY

10 wherein R 10 represents an alkanoyloxy or aroyloxy group is removed and the above group R 10 in the product obtained is converted, if desired, to a hydroxy group, or (b) a compound of formula I wherein R 1 represents hydroxy, alkanoylated or aroylated to prepare a hydroxy group. compound of formula I wherein R 1 represents alkanoyloxy or aroyloxy, or (c) if desired, a compound of formula I is converted to an amide at the 4-amino group with an alkanoyl or aroyl group.

Removal of the 3'-hydroxy group from a compound of formula II according to process (a) can be accomplished by first converting such a compound of formula II to the corresponding sulfonic acid ester such as the mesylate by treatment with

DK 165985B

3 is a sulphonic acid ehalide such as methanesulfonyl chloride, conveniently in the presence of an acid-binding agent, especially a tertiary amine such as pyridine, and at low temperature, e.g., 0-5 ° C. The sulfonic acid ester thus obtained can then be converted to the 3 'iodide in known manner, for example, by treatment with an alkali metal iodide such as sodium iodide in a suitable medium such as an aliphatic ketone, e.g. The resulting 3'-10 iodide can then be converted to a desired compound of formula I wherein R 1 is alkanoyloxy or aroyloxy by hydrogenation in the presence of a palladium catalyst. Alternatively, the above 3 'iodide may be treated with tributyltin hydride in the presence of a free radical initiator, e.g., azobisisobutyronitrile, conveniently in an inert organic solvent such as an aromatic hydrocarbon, e.g., benzene or toluene, and at elevated temperature, e.g. 60-90 ° C to give the desired compound of formula I wherein R 1 is alkanoyloxy or aroyloxy.

Another method of removing the 3'-hydroxy group from a compound of formula II comprises first reacting such a compound with phenylthionochloroformate, 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 or 4-dimethylaminopyridine, and at about room temperature to give a corresponding 3'-O-phenoxythiocarbonyl compound. Such a compound can then be converted to a desired compound of formula I, wherein R 1 is alkanoyloxy or aroyloxy, by treatment with tributyltin hydride in the presence of a free radical initiator analogous to that described above. In a preferred embodiment of Ises form of this process, the compound of Formula II is alkanoylated or aroylated at the 4-amino group prior to reaction with phenylthionochloroformate. In this embodiment, the product obtained after treatment with tributyltin hydride is a corresponding amide of a compound of formula I wherein R 1 is alkanoyloxy or aroyloxy.

DK 165985 B

4

The conversion of the alkanoylated or aroylated group into the product into a hydroxy group can be carried out in a manner known per se, for example by treatment with a saturated solution of ammonia in an alkanol, e.g. methanol. When the product carries an alkanoyl or aroylamino group at the 4-position, this group can simultaneously be converted to an amino group.

A compound of formula I wherein R 1 is hydroxy may be alkanoylated or aroylated according to process (b) to give a compound of formula I wherein R 1 is alkanoyloxy or aroyloxy. This esterification may be carried out in a manner known per se, for example, by treating the hydrochloride salt of a compound of formula I with a suitable acid halide in an inert organic solvent such as dimethylformamide.

The conversion of a compound of formula I into an amide according to process (c) can also be carried out in a manner known per se by treatment with a suitable alkanoylation or aroylating agent, respectively.

The starting materials of formula II can be prepared, for example, by conversion of the 3'-acyloxy group into a compound of the general formula III

NH2 * R220 where R10 is as defined above and R2 represents an acyl group to a 3 'hydroxy group.

DK 165985 B

5

This conversion can be carried out in known manner such as by treatment with ammonia or a suitable amine such as methylamine, ethylamine, n-propylamine or triethylamine in a lower alkanol such as methanol, conveniently at about 5 room temperature. It is to be understood that the reagent used to carry out the conversion as well as the reaction conditions should be selected in such a way as not to cause simultaneous conversion of the alkanoylated or aroylated group R10 to hydroxy.

The compounds of the above formula III are known compounds or analogous to known compounds which can be prepared in a similar manner to the known compounds.

The in vitro antiviral effect of the 1-cytosine derivatives of the present invention can be demonstrated in the experiments described below.

(A \ Effect against sheep lentivirus

This experiment uses sheep lentivirus (strain WLC-1) grown in sheep choroid plexus (SCP) cells using a medium containing 10% fetal bovine serum. The compounds are tested in 96 well microculture plates. Each well contains 104 SCP cells which have been incubated for 48 hours. Test compounds dissolved in dimethyl sulfoxide are added to a final concentration of 1% dimethyl sulfoxide together with a viral inoculum which is calculated to give 100% cytopathic effect in 12 days. After incubation at 37 ° C for 12 days, the plates are fixed and stained with crystal violet. The protection in wells containing test compounds is visually assessed against wells containing virus alone, and uninfected wells, the effect (MPC) is expressed as the lowest concentration. of test compound which provides 100% protection of the infected wells In this experiment DFAC has a MPC of 2.5 μΜ.

DK 165985 B

6

ΓΒΪ Effect against HIV

This experiment utilizes HTLV-III (strain RF) grown in C8166 cells (a human CD4 + T lymphoblastoid line) using RPMI 164O medium with bicarbonate buffer, antibiotics 5 and 10% fetal bovine serum. A suspension of cells is infected with 10 times the TCD50 of the virus and the adsorption is allowed to proceed for 90 minutes at 37 ° C. The cells are washed with medium.

The test is performed in 6 ml tissue culture glass, with each glass containing 2 x 10 5 infected cells in 1.5 ml medium. Test compound 10 dissolves either in aqueous medium or in dimethyl sulfoxide depending on solubility and a 15 μΐ solution of the substance is added. The cultures are incubated at 37 ° C for 72 hours in a humid atmosphere containing 5% carbon dioxide in air. The cultures are then centrifuged and an aliquot of the supernatant is solubilized and subjected to an "antigen capture technique" using a primary antiserum with particular reactivity to the viral protein 24 and a horseradish peroxidase detection system. The color formation is measured spectrophotometrically and plotted against Determine the concentration of test substance, the 20 concentration giving 50% protection (IC50).

In the experiment described above, DFAC has an IC5Q of 1 μΜ and the compound 1- (5'-O-benzoyl-2 ', 3'-dideoxy-2'-fluoro-0-D-arabinofuranosyl) cytosine has an IC5 , 1 μΜ.

The 1-cytosine derivatives of the present invention can be used as pharmaceuticals in the form of pharmaceutical compositions which are characterized in that they contain them together with a compatible pharmaceutical carrier. These compositions may be administered orally, for example in the form of tablets, dragees, hard gelatin capsules, soft gelatin capsules, solutions, emulsions or suspensions; or parenterally, for example in the form of injection solutions.

The above carrier may be a pharmaceutically inert, inorganic or organic carrier. Examples of such carriers which can be used for tablets, dragees and hard gelatine 7

DK 165985 B

capsules, are lactose, corn starch or derivatives thereof, talc, stearic acid or salts thereof. Examples of suitable carriers for soft gelatin capsules are vegetable oils, waxes, fats, semi-solid and liquid polyols. Suitable carriers for preparing solutions and syrups include, for example, water, polyols, sucrose, invert sugar and glucose. Suitable carriers for injection solutions are, for example, water, alcohols, polyols, glycerol and vegetable oils.

The pharmaceutical compositions may also contain conventional pharmaceutical adjuvants such as preservatives, solvents, stabilizers, wetting agents, emulsifiers, sweeteners, coloring or flavoring agents, salts for regulating the osmotic pressure, buffers, coatings or antioxidants. The pharmaceutical compositions may contain other therapeutically valuable substances.

The dose with which the 1-cytosine derivatives of the present invention can be administered varies depending on the strength of the derivative in question, the condition being treated, and the patient's needs as determined by the treating physician. In general, the 1-cytosine derivatives can be administered at a daily dose of about 0.1-20, preferably approx. 0.2-15 and in particular approx. 0.4-10 mg / kg body weight, although it is clear that these dose ranges are given by way of example only and may be varied upwards or downwards.

The aforementioned 1-cytosin derivatives may be administered in a single dose or preferably in multiple sub-doses, eg up to 6, distributed throughout the day. For example, a convenient unit dosage form for this administration may contain from about 0.5 to 300, preferably approx. 1.0 to 300 and especially about 2.0 to approx. 200 mg of 1-cytosine derivatives.

The pharmaceutical compositions can be prepared by mixing a 1-cytosine derivative mentioned above and, if desired, one or

DK 165985B

8 several other therapeutically valuable substances having a compatible pharmaceutical carrier and, if desired, a pharmaceutical adjuvant and bringing the mixture into a desired mode of administration. The preparation of pharmaceutical compositions can be carried out in a manner known per se.

The process of the invention is illustrated by the following examples.

EXAMPLE 1 a) A solution containing 0.94 g of 1-p'-O-acetyl-5'-O-10-benzoyl-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl) cytosine in 4.9 ml of a 2M solution of ammonia in methanol was stirred for 2 hours. The solution was evaporated to dryness and the residue was recrystallized from ethyl acetate to give 0.64 g of 1- (5'-O-benzoyl-2'-deoxy-2'-fluoro-β-D-arabino-furanosyl) cytosine, mp. 135-140 ° C.

b) A solution containing 0.4 g of the product of Example 1a) in 5.6 ml of dry pyridine was stirred at 0 ° C while 0.26 g of methanesulfonyl chloride was added dropwise. The mixture was stirred at 0-5 ° C for 20 hours and then treated with 0.1 ml of water. After stirring for 1 hour, the mixture was poured into 20 ml of ice / water and extracted with ethyl acetate. The ethyl acetate extracts were washed with saturated sodium chloride solution, dried over magnesium sulfate and evaporated to give 0.5 g of 1- (5'-o-benzoyl-2'-deoxy-2'-fluoro-3'-O-methylsulfonic acid). (N-β-D-arabinofuranosyl) cytosine.

c) A solution of 0.5 g of the product of Example 1b) and 0.9 g of sodium iodide in 12 ml of dry 2-butanone was stirred and refluxed for 16 hours. The resulting suspension was evaporated to dryness and the residue was partitioned between 20 ml of water and 12 ml of ethyl acetate. The phases were separated and the aqueous phase was extracted with ethyl acetate. The ethyl acetate solutions were dried over magnesium

DK 165985B

9 sulfate and evaporated. The residue was chromatographed on silica gel with methanol / dichloromethane (1: 9). This gave 0.2 g of 1- (5 / O-benzoyl-2 ', 3'-dideoxy-2'-fluoro-3'-iodo- / 9-D-arabino-furanosyl) cytosine.

D) A solution of 0.2 g of the product of Example 1c) in 8 ml of ethanol was treated with a solution of 0.04 g of sodium bicarbonate in 2 ml of water and then hydrogenated in the presence of 0.06 g of 10% palladium on -carbon catalyst for 24 hours. The catalyst was removed by filtration and the filtrate was evaporated to dryness. The residue was partitioned between water and ethyl acetate. The aqueous phase was separated and back extracted with ethyl acetate. The ethyl acetate solutions were dried over magnesium sulfate and evaporated.

This gave 0.14 g of 1- (5'-O-benzoyl-2 ', 3'-dideoxy-2'-fluoro-β-15D-arabinofuranosyl) cytosine.

e) 0.05 g of the product of Example 1d) was dissolved in 2 ml of a saturated solution of ammonia in methanol. The solution was allowed to stand for 3 days and then evaporated to dryness. The residue was dissolved in 2 ml of water and the solution 20 was washed with ethyl acetate. The aqueous solution was lyophilized for 24 hours to give 0.02 g of DFAC, MS (EI): m / e 229 (M) +, 151, 112.

Example 2 a) A solution containing 11.8 g of the product of Example 1a) in 1180 ml of methanol was stirred and heated to reflux. The solution was treated with 12 ml of acetic anhydride and then at 1 hour intervals for an additional 4 hours with each time 12 ml of acetic anhydride. After 6 hours, the solution was evaporated to dryness and the residue was co-evaporated twice with 300 ml of toluene each time and then redissolved in 1 liter of methanol and treated at 1 hour intervals over 3 hours with 12 ml of acetic anhydride each time. . The solution was

DK 165985B

The mixture was evaporated to dryness and the residue dissolved in dichloromethane. the solution was washed with water, saturated sodium hydrogen carbonate solution and water and then dried over sodium sulfate and evaporated. The residue was dried in vacuo to give 12.5 g of N-acetyl-1- (5'-O-benzoyl-2'-deoxy-2'-fluoro-9-D-arabinofuranosyl) cytosine.

b) A solution containing 10.7 g of the product of Example 2a) and 29.7 g of 4- (dimethylamino) pyridine in 295 ml of acetonitrile was stirred under an argon atmosphere and cooled to 10 5 ° C while 5.61 g of phenyl chlorothionocarbonate was added. added drip proof. The mixture was stirred at 5 ° C for a further 10 minutes and then at room temperature for 3 hours. The resulting solution was evaporated to dryness and the residue dissolved in dichloromethane. The solution was washed with 15 ice-cold water, 1M hydrochloric acid, water, saturated sodium hydrogen carbonate solution and saturated sodium chloride solution, and then dried over sodium sulfate and evaporated. The residue was dried in vacuo to give 14.2 g of N-acetyl-1- (5'-o-benzoyl-2 '-deoxy-2' -fluoro-3 '-O-phenoxythiocarbony 1-y3-D-2 (Arabinofuranosyl) cytosine.

c) A solution containing 14.1 g of the product of Example 2b) and 0.5 g of azobisisobutyronitrile in 520 ml of dry toluene was stirred while argon atmosphere was bubbled through the solution for 10 minutes. 11.28 g of tributyltin hydride was added and the introduction of argon atmosphere was continued for a further 30 minutes. The mixture was then heated at 75 ° C under argon atmosphere for 3 hours. The solution was evaporated to dryness and the residue was triturated with hexane. The product was filtered off and purified by chromatography on silica gel with methanol / dichloromethane (1: 9).

This gave 6.95 g of N-acetyl-1- (5'-O-benzoyl-2 ', 3'-dideoxy-2'-fluoro-β-D-arabinofuranosyl) cytosine, m.p. 191-193.5 ° C, after recrystallization from toluene. (D) A solution containing 0.5 g of the product of Example 2c) in 50 ml of methanol which had already been obtained

DK 165985 B

saturated with ammonia at 0 ° C, was stirred for 3 days. The solution was evaporated to dryness and the residue was dissolved in 30 ml of water. The aqueous solution was washed with ethyl acetate and then evaporated to dryness. The residue was recrystallized from ethanol to give 0.18 g of DFAC, m.p. 204-207 "C. The mother liquors from the recrystallization were evaporated and the residue dissolved in distilled water. The aqueous solution was lyophilized and the residue was recrystallized from ethanol to give an additional yield of 0.06 g of DFAC, m.p. 199-203 "C.

The following examples illustrate typical pharmaceutical compositions containing as the active ingredient the 1-cytosine derivatives of the present invention.

EXAMPLE A

Conventionally, tablets can be prepared containing the following ingredients:

Ingredients Pr. tablet

Active ingredient 10 mg

Lactose 20 mg 20 Starch 4 mg

Polyvinylpyrrolidone 0.5 mg

Magnesium stearate 0.5 mo

Tablet weight 35 mer

EXAMPLE B

25 Ingredients Pr. capsule

Active ingredient 25 mg

Lactose 15 mg

Sodium starch glycollate 2.5 mg

Magnesium stearate 0.5 ma 30 Capsule filling weight 43 ma

Claims (4)

A compound according to claim 1, 10, characterized in that it is 1- (2 ', 3'-dideoxy-2'-fluoro- / 9-D-arabinofuranosyl) cytosine. A 1-Cytosine derivative according to claim 1 or 2 for use as a therapeutic active substance, especially for use in the treatment or prophylaxis of viral infections, especially retroviral infections, especially HIV infections. Process for the preparation of 1-cytosine derivatives according to claim 1, characterized in that the (a) 3 'hydroxy group in a compound of the general formula II wherein R10 represents an alkanoyloxy or aroyloxy group , 5 is removed and the above group r 1 ° of the product obtained is converted into a hydroxy group if desired, or (b) a compound of formula I wherein R 1 is hydroxy, alkanoylated or aroylated to prepare a compound of formula I wherein R 1 is alkanoyloxy or aroyloxy, or (c) if desired, a compound of formula I is converted to an amide at the 4-amino group with an alkanoyl or aroyl group. Medicament, especially for the treatment or prophylaxis of viral infections, especially retroviral infections, especially HIV infections, characterized in that it contains an I-cytosine derivative according to claim 1 or 2 and a compatible pharmaceutical carrier. Use of a 1-cytosine derivative according to claim 1 or 2 for the manufacture of a medicament for the treatment or prophylaxis of viral infections, especially retroviral infections, especially HIV infections.