NO177306B - Analogous Process for the Preparation of Therapeutically Active Purine Derivatives - Google Patents

Description

The present invention relates to a method for the production of new chemical compounds and pharmaceutically acceptable salts thereof for therapeutic use in the treatment and prevention of AIDS (Acquired Immuno Deficiency Syndrome) and infections caused by viruses that require reverse transcriptase for replication, such as HIV (human immunodeficiency viruses) and hepatitis B virus types, as well as for the treatment of other viral diseases, e.g. caused by the herpes virus, diseases that include both common infections and neoplastic diseases, i.e. cancer.

Background for the invention

The effects of viruses on bodily functions are the end result of changes occurring at the cellular and subcellular levels. The pathogenic changes at the cellular level are different for different combinations of virus and host cells. While some viruses cause a general destruction (killing) of some cells, others can transform cells into a neoplastic state.

Important common viral infections are herpes dermatitis (including herpes labialis), herpes keratitis, herpes genitalis, herpes zoster, herpes encephalitis, infectious mononucleosis and cytomegalovirus infections, all of which are caused by viruses belonging to the herpes virus group. Other important viral diseases are influenza A and B, which are caused by influenza A and B viruses respectively. Another important viral disease is viral hepatitis, and hepatitis B virus infections in particular are very widespread. There is a need for effective and selective antiviral agents for the treatment of these diseases as well as other diseases caused by viruses. Several different viruses of both DNA and RNA type have been shown to cause tumor formation in animals. In animals, the effect of carcinogenic chemicals can result in the activation of latent tumor viruses. It is possible that tumor viruses are involved in human tumors. Currently, the most likely human cases are leukaemias, sarcomas, breast carcinomas, "Burkitt lymphomas", nasopharyngeal carcinomas and cervical cancers where RNA tumor viruses and herpes viruses are included. This makes research into selective inhibitors of tumorigenic viruses and their functions an important field in the treatment efforts for cancer.

In the latter part of the 70s, a new disease was reported which was later referred to as AIDS (Acquired Immuno Deficiency Syndrome). It is now generally accepted that a retrovirus called HIV (Human Immunodeficiency Virus), formerly known as human T-cell lymphotropic virus (HTLV-III) or LAV (Lymphadenopathy Associated Virus) plays a significant role in AIDS etiology. Different types of HIV have been found, such as HIV-1 and HIV-2, and more can probably be expected to be isolated.

AIDS is characterized by a profound immunodeficiency resulting from a small number of a subset of lymphocyte T-helper cells, which are a target of attack in HIV infection. The profound immunodeficiency in AIDS patients makes these patients very vulnerable to a number of opportunistic infections of bacterial, fungal, protozoal or viral etiology. The etiological agents among viral opportunistic infections are often found in the herpes group, i.e. herpes simplex virus (HSV), Varicella Zoster virus (VZV), Epstein-Barr virus (EBV) and especially cytomegalovirus (CMV). Other retroviruses that attack animals are feline leukemia virus and equine infectious anemia virus. Human diseases such as multiple sclerosis, psoriasis and Kawasaki disease have also been reported to be linked to retrovirus infections.

Hepatitis B virus infections cause serious diseases such as acute hepatitis, chronic hepatitis, fulminant hepatitis in a significant number of people. It has been estimated that there are 200 million patients with chronic hepatitis B infection in the world. A significant number of the chronic cases progress to liver cirrhosis and liver tumors. In some cases, the hepatitis infections also assume a rapid and serious course, as in fulminant hepatitis B with approx. 90% mortality. Currently, there are no known effective treatments for hepatitis B infections. Hepatitis B virus replication is similar to that of retroviruses and contains the same essential viral reverse transcriptase activity

General description of the invention

A large number of nucleoside analogues exhibit multiple anti-metabolic effects. They do this by replacing, or by competing with, the naturally occurring nucleosides. Recently, some nucleoside analogues have been described which in cell culture inhibit the propagation of HIV (also called HTLV-III, LAV) the disease-causing agent of AIDS and AIDS-related complex (ARC).

We have now discovered that inhibitory effects of HIV and/or herpes propagation are exhibited by nucleoside analogues where the nucleoside bases are both natural and modified purine bases which, in the N-9 position, are derivatized with an acyclic side chain, which is

branched in the 2' position and contains functional groups.

Previous acquaintance

Purine derivatives with antiviral action have previously been discussed in the following references:

9-(phosphonylmethoxyalkyl)adenines are described in AU-A-56328/86 and AU-A-56468/86;

9-(1,3-dihydroxy-2-propoxymethyl)purines and cyclic phosphate esters are described in US-A-4,565,868, US-A-4,590,269 and EP-A-184,473; and

9-(4-hydroxy-3-hydroxymethylbutyl)purine derivatives are described in EP-A-141 927.

Moreover, the compound of formula is known from EP-A-186 64 0; and the compounds with formula

where n=1 or 2, known from EP-A-146 516.

Description of the invention

According to the present invention, it has now been shown that the compounds with formula

where:

R<1> is hydrogen, hydroxy, mercapto or amino; R<2> is hydrogen, hydroxy, fluorine, chlorine or amino; R<3> and R<4>, independently of each other, are selected from

hydroxy or an ester residue thereof that writes

from a carboxylic acid R<6>COOH, where R<6> is hydrogen, C1~6-alkyl or phenyl, or

R<3> together with R<4> are

where

M is hydrogen, C1-C4 alkyl or a pharmaceutically acceptable counterion; and n is 1 or 2; with the proviso that when R<3> and R<4> are hydroxy, then R<1> is not hydrogen, hydroxy or chlorine, and pharmaceutically acceptable salts thereof,

inhibits the multiplication of HIV. The compounds of formula I can be used as therapeutic and/or prophylactic agents in the control and treatment of human HIV virus infections.

According to a more general aspect, the compounds of formula I are suitable as therapeutic and/or prophylactic agents in the control and treatment of infections caused by retroviruses and hepatitis B viruses in mammals including humans.

All retroviruses, including HIV, require the enzyme reverse transcriptase in their natural replication cycle.

Hepatitis B virus (HBV) is a DNA virus with a unique ring-shaped double-stranded DNA genome that is partially single-stranded. It contains a specific DNA polymerase needed for viral replication. This DNA polymerase also serves as a reverse transcriptase during the replication of HBV DNA via an RNA intermediate.

The compound of formula I inhibits the activity of reverse transcriptase in retroviruses, including HIV, as well as the activity of DNA polymerase in hepatitis B virus.

Another important area of use of the compounds of formula I is in the treatment of herpes virus infections. Among the various herpes viruses, herpes simplex type 1 and 2, varicella (Herpes Zoster), viruses that cause infectious mononucleosis (ie Epstein-Barr virus) and cytomegalovirus can be mentioned. Important diseases caused by herpes viruses are herpes dermatitis (including herpes labialis), herpes genitalis, herpes keratitis, herpes encephalitis and herpes zoster.

Another possible area of use for the compounds according to the invention is the treatment of cancer and tumors, especially those caused by viruses. This effect can be achieved in different ways, ie by inhibiting the transformation of virus-infected cells into a neoplastic state, by inhibiting the spread of virus from transformed cells to other normal cells and by arresting the growth of virus-transformed cells.

The compounds of the above general formula I can be used for the preparation of a drug for the therapeutic and/or prophylactic treatment of AIDS, as well as infections caused by viruses that require reverse transcriptase for their replication.

Preferably, they can be used for the treatment of infections caused by the HIV virus or the hepatitis B virus.

The compounds of formula I contain one asymmetric center when CH2CH2R<3> and (CH2)nR<4> are different. They therefore occur in two optical forms which constitute a further aspect of the invention.

Preferred compounds for use according to the invention are those where R<1> and R<2>, independently of each other, are hydrogen, hydroxy or amino and where R<3> is

hydroxy or an ester derivative thereof which is written from a carboxylic acid R<6>COOH, where R<6> is hydrogen, C-6 alkyl or phenyl, and R<4> is OH or an ester derivative thereof, or where R<3 > and R<4> together are

Preferably both R<3> and R<4> are hydroxy; with the proviso that when R<3> and R<4> are hydroxy, then R<1> cannot be hydrogen, hydroxy or chlorine.

Examples of particularly preferred compounds are those of formula I

It is understood that the examples of pharmaceutically acceptable salts mentioned below also apply to the various esters or derivatives of the purine bases produced according to the invention.

Examples of pharmaceutically acceptable salts of . the compounds of formula I include base salts, e.g. obtained from a suitable base such as an alkali metal (eg sodium, potassium), alkaline earth metal (eg magnesium) salts, ammonium and NX4~ (where X is C1_4 alkyl). Physiologically acceptable acid salts include salts of organic carboxylic acids such as acetic acid, lactic acid, gluconic acid, citric acid, tartaric acid, maleic acid, malic acid, pantothenic acid, isethionic acid (2-hydroxyethanesulfonic acid), oxalic acid, lactobionic acid and succinic acid; organic sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid and p-toluenesulfonic acid, as well as inorganic acids such as hydrochloric acid, hydrogen iodide, sulfuric, phosphoric and sulfamic acids.

Physiologically acceptable counterions for the phosphonate groups include inorganic and organic counterions. Inorganic sports ions are, for example, ammonium, sodium, potassium, lithium, magnesium and calcium. Organic counterions are obtained from non-toxic bases, such as primary, secondary and tertiary amines, including naturally occurring amines. Examples of such amines are diethylamine, triethylamine, isopropylamine, ethanolamine, morpholine, 2-diethylaminoethanol, glucosamine, N-methylglucamine, piperazine and dicyclohexylamine.

The present invention relates to a method for the production of new compounds with formula

where:

R<1> is hydrogen, hydroxy, mercapto or amino;

R<2> is hydrogen, hydroxy, fluorine, chlorine or amino;

R<3> and R<4>, independently of each other, are selected from

hydroxy or an ester residue thereof which is written from a carboxylic acid R<6>COOH, where R<6> is hydrogen, C^-g-alkyl or phenyl, or R<3> together with R<4> is

, where

M is hydrogen, C1-C4 alkyl or a pharmaceutically acceptable counterion; and n is 1 or 2; with the proviso that when R<3> and R<4> are hydroxy, then R<1> is not hydrogen, hydroxy or chlorine, and pharmaceutically acceptable salts thereof.

A pharmaceutical preparation may comprise a new compound with formula I as active ingredient; and can be used for the therapeutic and/or prophylactic treatment of viral infections in an animal or in humans in need of treatment, which consists in providing an effective amount of a new compound of formula I.

In particular, it is relevant to treat infections caused by herpes virus or a virus that requires reverse transcriptase for replication, including human immunodeficiency virus and hepatitis B virus.

In clinical use, the purine derivatives of formula I will normally be administered orally, by injection or by infusion in the form of a pharmaceutical preparation comprising the active ingredient in the form of the original compound, or possibly in the form of a pharmaceutically acceptable carrier which can be a solid, half - solid or liquid diluent or a swallowable capsule. The compound can also be used without carrier material. Examples of pharmaceutical preparations include tablets, dragees, capsules, granules, suspensions, mixtures, syrups, solutions, etc. Usually the active substance will make up between 0.05 and 20% in preparations intended for injection, and between 10 and 90% for preparations intended for oral administration.

When treating patients suffering from retrovirus, especially HIV or hepatitis B virus infections, it is preferred to administer the compound orally, parenterally, rectally, nasally, topically or vaginally. The parenteral administration includes subcutaneous, intramuscular, intravenous and sublingual administration. Local administration includes buccal and sublingual administration. The dosage of the active substances can vary within a wide range and will depend on various factors such as the degree of the infection, the patient's age, etc., and may have to be adapted individually. A suitable range for the amount of the compounds according to the invention, or a physiologically acceptable salt thereof, for daily administration may be from approx. 10 mg to 10,000 mg, preferably 100-500 mg for intravenous administration and preferably 100-3000 mg for oral administration.

Compounds of formula I can act synergistically or additively with a wide variety of other therapeutic agents, whereby the therapeutic potential of both agents is increased without increasing the toxic effects, so that the therapeutic ratio is increased.

A compound of formula I or a pharmaceutically acceptable derivative thereof can be used in combination therapy, where the two active substances occur in a ratio that results in an optimal therapeutic ratio. This can be achieved either by a synergistic effect against the viral infection, and/or by lowering the toxicity while maintaining an additive or synergistic therapeutic effect.

The optimal therapeutic ratio is observed when the two agents occur in a ratio of 500:1 to 1:500, preferably 100:1 to 1:100, especially 20:1 to 1:20 and especially 10:1 to 1:10.

The combinations may conveniently be administered together, for example in a unitary pharmaceutical formulation, or separately, for example as a combination of tablets and injections given simultaneously or at different times, to achieve the required therapeutic effect.

The compounds of formula I are potentiated by interferons, other antiviral agents, such as foscarnet, AZT, HIV protease inhibitors, immunomodulators, interferon-inducing agents and growth factors.

Particularly preferred types of interferon are a, /? and interferon-inducing agents such as "Ampligen" (Hem Research).

Other combinations suitable for use according to the present invention include those where the second agent is, for example, interleukin II, suramin, foscarnet or an ester thereof, HPA 23, inhibitors of HIV protease, such as pepstatin, steroids, drugs such as levamisole or thymosin to increase lymphocyte numbers and/or function, or GM-CSF and other factors that regulate cell functions.

Manufacturing methods

According to the invention, the compounds of formula I can be prepared by one of the following general methods:

A. Condensation of an acyclic side chain compound as in formula I, to the N-9 position of a purine derivative. The acyclic side chain has a terminal leaving group and the functional groups can optionally be protected with known groups used for the protection of hydroxy, amino or phosphonate functions.

Examples of suitable derivatives of the reactants are those where R<1>' is Cl, or R1 as defined above, R<1>, R<2>, R3, R<4> and n are as defined above and W is an appropriate leaving group such as Cl, Br, I, alkyl or arylsulfonyloxy, trifluoromethanesulfonyloxy. The condensation reaction is carried out in an organic solvent such as dimethylformamide, dimethylsulfoxide, ethanol, acetonitrile, dichloromethane or the like, at a temperature of between 0°C and 150°C for a period of time from 1 hour to 5 days, and after the condensation the products can be hydrolysed or are converted by known conventional methods into compounds of formula I.

In the case of a phosphonate, the side chain, condensed to a purine base, can be prepared in different ways. An example is the following reaction sequence, where the starting material 5-(2-bromomethyl)-2,2-dimethyl-1,3-dioxane is described (M.R. Harnden & R.L. Jarvest, Tetrahedron Letters, Vol. 26, pp. 4265-4268, 1985).

a) P(OMe)3; b) H + , MeOH; c) MeO-; d) N-bromosuccinimide, triphenylphosphine; A compound of formula I where R<3> and R<4> together are can be subjected to ring cleavage to form a compound wherein one of R<3> and R<4> is

and another is hydroxy.

The method described above can be used to give mixtures of optical isomers or, in appropriate situations, a single optical isomer. A compound according to the invention in the form of an optical isomer can be produced if, in the aforementioned method, an optically active acyclic side chain is condensed to the N-9 position in the purine derivative, or if the condensation is directed towards the formation of an optical isomer using another optically active connection. A single optical isomer can also be obtained from the racemic mixtures by methods known per se.

The following examples will further illustrate the invention.

Example 1

2-(2-aminopurin-9-yl)methylbutane-1.4-diol- diacetate

A mixture of 4-acetoxy-2-bromomethylbutyl acetate

(0.465 g, 1.74 mmol), 2-aminopurine (0.282 g, 2.09 mmol) and powdered potassium carbonate (1.20 g, 8.70 mmol) in N,N-dimethylformamide (20 mL) were stirred at room temperature for 5 days. Chloroform (40 mL) was added, solids removed by filtration and the solution evaporated in vacuo to a small volume. Chromatography on 50 g SiO2 with chloroform + methanol (7 + 1) as eluent gave a fraction of 70-130 ml which was evaporated and dried in vacuo, finally under 0.1 mBar to give 0.349 g (62%) 2-( 2-Aminopurin-9-yl)methylbutane-1,4-diol diacetate. TLC on silica (chloroform + methanol, 7+1): Rf 0.57.

<X>H NMR (CDCl 3 + CD 3 OD) : S 8.64 (s, 1H), H 6 ; 7.92 (s, 1H) H8 ; 5.82 (broad s, 2H) NH 2 ; 4.3-4.15 (m, 4H) 2 CH 2 OAc; 4.33 (d, 2H) CH 2 N; 2.50 (m, 1H) CH; 2.06 (s, 6H) 2 CH 3 COO; 1.75 (q, 2H) CH 2 CH 2 OAc;

<13>C NMR (CDCl3<+> CD3OD): S 170.96, 170.66 (2 C=0);

159.89 (C2); 153.13 (C4); 148.77 (C6); 142.84 (C8); 126.83 (C5); 63.78 (CHCH 2 -OAc); 61.47 (CH 2 CH 2 OAc); 44.05 (CH 2 N); 35.15 (CH) ; 27.59 (CH 2 CH 2 O); 20.22, 20.05 (2CH3) . The starting materials were prepared according to the following reaction sequence (a-e):

a) a- trityloxymethyl-" Y- butyrolactone

A mixture of α-hydroxymethyl-β-butyrolactone (26.83 g,

0.231 mol) (G. Claeson et H. G. Jonsson, Arkiv for Kemi 28, 167 (1967)), trityl chloride (77.3 g, 0.277 mol) and dry pyridine (200 ml) were stirred at room temperature for a few hours until it was homogeneous. After 10 days at room temperature, the solution was poured into a mixture of 500 ml of water and 500 ml of n-hexane. The precipitate was washed with water and hexane and finally dried under 0.1 mBar to give 65.60 g (79%) of crude product which was contaminated with some trityl alcohol. TLC on silica (ethyl acetate + n-hexane (1+3)): Rf 0.38.

<13>C NMR (CDCl3): S 177.84 (C=O); 143.71, 128.68, 127.96 and 127.20 (phenyl); 86.96 (0 CPH3); 67.26 (CH 2 0 CO); 62.49 (CH 2 OTr ); 40.31 (CH) ; 26.15 (CH 2 CH 2 O) .

b) 2-trityloxymethyl-1,4-butanediol

α-Trityloxymethyl-'Y-butyrolactone (60.21 g, 0.168 mol) was

in small portions added to a stirred suspension of lithium aluminum hydride (9.53 g, 0.251 mol) in dry tetrahydrofuran (300 mL), after which the mixture was refluxed for 1 hour. Slow addition of 10 ml H 2 O + 10 ml 15% NaOH and 30 ml H 2 O led to a white sandy precipitate, which was filtered off and washed with 2 x 50 ml tetrahydrofuran. The filtrate was evaporated to a small volume and dissolved in diethyl ether (300 ml), added silica gel (250 g) and carefully evaporated to a homogeneous powder. This crude product-silica gel mixture was placed over a layer of silica gel (250 g) in n-hexane in a chromatography column. Elution with ethyl acetate + n-hexane (1+3), 2200 ml, removed the trityl alcohol. Continued elution with ethyl acetate + ethanol (9+1) gave fractions 2900-3700 (from the start) which after evaporation in vacuum led to a crystallizing oil. Yield 52.77 g (87%). TLC on silica gel: ethyl acetate + n-hexane (1+3), Rf 0.05; ethyl acetate + ethanol (9+1), Rf 0.81.

c) 2-trityloxymethyl-1,4-butanediol diacetate

To a stirred mixture of 2-trityloxymethyl-1,4-butanediol

(50.85 g, 0.140 mol) and triethylamine (42.6 g, 0.42 mol) in dry diethyl ether (500 mL) was slowly added a solution of acetyl chloride (27.5 g, 0.35 mol) in ether (25 ml) under external cooling with cold water to keep the mixture at room temperature. After 45 minutes, the triethylamine hydrochloride was filtered off and washed with a little ether. The combined filtrates were washed with water (50 mL), 0.5M hydrochloric acid (100 mL) and water (50 mL), dried over magnesium sulfate and evaporated in vacuo, finally at 0.1 mBar to give 61.03 g (97 %) oily crude product. TLC on silica (ethyl acetate + n-hexane 1+1): Rf 0.68.

d) 4-acetoxy-2-hydroxymethylbutyl acetate

2-trityloxymethyl-1,4-butanediol diacetate (60.90 g,

0.136 mol) was dissolved in acetic acid (320 ml) at 100°C and water (80 ml) added. The solution was held at 100°C for 15 min., evaporated in vacuo to a small volume and cooled to 0°C.

The precipitate was filtered off and washed with cold ethyl acetate to give 26.44 g (35.51 g of theory) of trityl alcohol. The combined filtrates were evaporated to a small volume. The compound was purified on a silica gel column (500 g SiO 2 ); eluent 0-2700 ml ethyl acetate + n-hexane (1+1), 2700-3740 ml ethyl acetate + n-hexane (2+1) and then undiluted ethyl acetate. Fractions 2540-4800 ml were evaporated to give 14.20 g (51%) of pure 4-acetoxy-2-hydroxy-methylbutyl acetate. TLC on silica (ethyl acetate + n-hexane 1+1): Rf 0.30.

<13>C NMR (CDCl3 ): S 171.08, 170.88 (2 C=0); 64.07 (CH 2 OH); 62.10, 61.45 (2 CH 2 OAc); 37.07 (CH) ; 26.76 (CH 2 CH 2 OAc); 20.39 (2CH3).

e) 4-acetoxy-2-bromomethylbutyl acetate

A solution of 4-acetoxy-2-hydroxymethylbutyl acetate

(11.04 g, 0.054 mol) and triphenylphosphine (21.27 g, 0.081 mol) in dry dichloromethane (150 mL) were stirred at 0°C and N-bromosuccinimide (14.43 g, 0.081 mol) added portionwise. The mixture was kept at 0°C for 20 hours, evaporated to a small volume and stirred with 50 ml of ethyl acetate + n-hexane (1+1). The white triphenylphosphine oxide was filtered off and washed with a little ethyl acetate + n-hexane (1+1). The combined filtrates were evaporated and purified on a 200 g SiO 2 column with ethyl acetate + n-hexane (1+1) as eluent. Fractions 250-550 ml were evaporated in vacuo to give 11.90 g (82%) of pure 4-acetoxy-2-bromomethylbutyl acetate. TLC on silica (ethyl acetate + n-hexane 1+1): Rf 0.59.

<2>H NMR (CDCl3) : <5 4.2-4.0 (m, 4H), 2 CH2OAc; 3.53 (ABX system, 2H) CH 2 Br; 2.25-2.1 (m, 1H) CH; 2.08, 2.06 (2 s, 2x3H) 2 COCH 3 ;

1.79 (m, 2H) CH 2 CH 2 OAc.

<13>C NMR (CDCl3): δ 170.91, 170.74 (2 C=0); 64.90 (CHCH 2 0 Ac); 61.71 (CH 2 CH 2 OAc); 36.56 (CH); 34.74 (CH 2 Br); 28.88 (CH 2 CH 2 O); 20.95 (2CH3).

Example 2 9-(4-acetoxy-2-acetoxymethylbutyl)guanine-f 2-(guanine-9-ylmethyl)-1,4-butanediol. diacetate]

A mixture of 9-(4-hydroxy-2-hydroxymethylbutyl)guanine (0.50 g, 2.0 mmol), acetic anhydride (1.02 g, 10.0 mmol), pyridine (1.11 g, 14.0 mmol) and dry N,N-dimethylformamide (25 mL) were stirred at room temperature for 13 days and then evaporated to dryness in vacuo. The crystalline residue was heated with 10 mL of water and lyophilized and recrystallized from water to give 0.468 g (69%) of 9-(4-acetoxy-2-acetoxymethylbutyl)guanine.

<13>C NMR (CDCl3+CD3OD): S 64.15 (CHCH2O); 61.98 (CH 2 CH 2 O); 44.71 (CH2N); 35.88 (CH) ; 27.95 (CH 2 CH 2 O); 20.87, 20.68 (2CH3)

Example 3 9-(4-propionoxy-2-propionoxymethylbutyl)guanine f 2-(guanine-9-ylmethyl)-1,4-butanediol dipropionate1

A mixture of 9-(4-hydroxy-2-hydroxymethylbutyl)guanine (0.50 g, 2.0 mmol), propionic anhydride (1.56 g, 12.0 mmol), pyridine (1.27 g, 16.0 mmol) and dry N,N-dimethylformamide (25 mL) were stirred at room temperature for 14 days and then evaporated to dryness in vacuo. The crystalline residue was heated with 10 mL of water and lyophilized and recrystallized from water to give 0.418 g (57%) of 9-(4-propionoxy-2-propionoxymethylbutyl)-guanine.

< 13>C NMR (CDCl 3 +CD 3 OD) : S 64.00 (CHCH 2 O); 61.86 CH 2 CH 2 O);

44.78 (CH 2 N); 35.95 (CH); 28.00 (CH 2 CH 2 O); 27.56,

27.44 (2C<H>3CH2CO); 9.00 (2CH3).

Example 4 9-(4-hydroxy-2-hydroxymethylbutyl)adenine

Dimethyl 2-(adenin-9-ylmethyl)succinate (2.93 g, 0.010 mol) was dissolved under heating in tert. butanol (120 ml), lithium borohydride (1.10 g, 0.05 mol) added portionwise and the mixture stirred at room temperature for 3 hours, water (10 ml) added and stirring continued overnight. Inorganic material was filtered off and washed with tert. butanol, whereupon the filtrate was evaporated to a small volume. Chromatography on silica (ethyl acetate + methanol + water 7+2+1) led to pure 9-(4-hydroxy-2-hydroxymethylbutyl)adenine.

<13>C NMR (DMSO-d 6 ): δ 156.24 (C 6 ); 152.67 (C2); 150.14 (C4); 141.84 (C8); 118.88 (C5); 61.18, 58.92 (2 CH 2 OH); 44.81 (CH 2 N); 38.36 (CH); 32.02 (CH2C<H>2OH).

The starting material was prepared as follows:

Dimethyl 2-(adenin-9-ylmethyl)-succinate

A mixture of adenine (5.40 g, 0.040 mol), dimethyl itaconate (8.00 g, 0.051 mol), sodium hydride (55% in oil, 0.2 g) and dry N,N-dimethylformamide (125 mL) was heated to 120°C and then kept under stirring at room temperature for 8 days. The precipitate was filtered off, washed with dichloromethane (3x15 mL) and dried in vacuo to give 8.96 g (76%) of dimethyl 2-(adenin-9-ylmethyl)-succinate.

1 H NMR (CDCl 3 ); S 8.28 (s, 1H) H 2 ; 7.90 (s, 1H) H8 ;

4.54 (ABX system 2H) CH2N; 3.70, 3.69 (2s, 2x3H) 0CH3;

3.46 (m, 1H) CH; 2.72 (d, 2H)CH2CO0.

<13>C NMR (CDCl3+CD3OD): S 172.39, 171.42 (2 C=O); 155.61 (C6); 152.91 (C2); 149.87 (C4); 141.01 (C8); 118.85 (C5); 52.37, 51.94 (OCH 3 ); 44.10 (CH 2 N); 41.55 (CH); 33.30 (CH 2 COO).

Example 5 Sodium ethyl 3-(quanin-9-ylmethyl)-4-hydroxybutanephosphonate and 7-isomer

2-amino-6-chloro-9-[(2-ethoxy-2-oxo-1,2-oxaphosphorinan-5-yl)-methyl]purine (VSC 655) and its 7-isomer (100 mg,

0.29 mmol), dissolved in ethanol (4 mL), water (4 mL) and 2M aqueous sodium hydroxide (0.90 mmol) was kept at 37°C for 18 hours. The solution was neutralized by the addition of weakly acidic Amberlite cation exchange resin, filtered and evaporated to

dryness to give 113 mg (quantitative yield) of crude product.

• 1 H NMR (D 2 O, tert. BuOH, 200 MHz): S 8.01 and 7.79 (s, 8H, 7 and 9 isomers); 4.03 (m, CH 2 N); 3.78 (quintet, CH2QP); 3.50 (d, CH 2 OH); 2.05 and 1.6-1.3 (m, PCH2CH2CH); 1.12 (t, CH 3 C-O-P) . <13>C NMR (D 2 O, tert. BuOH, 50 MHz): S 161.81, 160.47, 154.12, 145.5, 142.13, 114.55, 61.74/61.42 (CH 2 OP ); 45.35 and 45.15 (CH 2 N); 42.25/41.91 (CH) ; 25.59, 22.89; 16.83 (CH 3 C-O-P) . Example 6 Disodium 3-(guanin-9-ylmethyl)-4- hydroxybutane phosphonate

A solution of 2-amino-6-chloro-9-[(2-ethoxy-2-oxo-1,2-oxaphosphorinan-5-yl)methyl]purine (VSC 655; 102 mg, 0.295 mmol) in ethanol (2 ml), water (2 ml) and 2M aqueous sodium hydroxide (1.0 ml, 2 mmol) were kept at 80°C for 3 days, neutralized by addition of weakly acidic Amberlite cation exchange resin, filtered and evaporated to dryness to give disodium 3-(guanin-9-ylmethyl)-4-hydroxybutanephosphonate.

The starting materials for Examples 5 and 6 were prepared as follows (including example 7):

2-(acetoxymethyl)-4-bromobutyl acetate

This intermediate was synthesized from 4-(acetoxy)-3-(acetoxymethyl)-butanol according to methods described in the literature. Yield 97% after flash column chromatography on silica (ethyl acetate + n-hexane 1+1).

TLC Rf 0.67 (SiO 2 , ethyl acetate + n-hexane 1+1).

<13>C NMR (CDCl3, TMS, 50 MHz): S 170.30 (COO); 63.44 (CH 2 O); 36.27 (CH); 31.67 (Br-CH 2 ); 30.29 (Br-C-CH 2 ); 20.51 (CH 3 ).

Diethyl 4-acetoxy-3-(acetoxymethyl)butanephosphonate

Triethyl phosphite (2.70 g, 16.3 mmol) was added to 2-(acetoxymethyl)-4-bromobutyl acetate (VSC 647,

3.95 g, 14.8 mmol) at 180-190°C and stirring continued at 190°C for 0.5 hours. The residue was evaporated in vacuum and kept below about 0.1 mBar. Flash chromatography on silica with ethyl acetate + ethanol (9+1) gave 3.36 g (70%) of the product.

TLC Rf 0.57 (SiO 2 , ethyl acetate + ethanol 9+1).

<13>C NMR (CDCl3, TMSD, 50 MHz): δ 170.37 (COO); 63.22 (CH 2 0 Ac); 61.30/61.18 (CH 2 OP, J 6 Hz); 37.58/37.27 (CH, J 16 Hz); 24.11/21.29 (CH2-C-P, J 142 Hz); 20.97/20.90 (CH 2 P J 4 Hz); 20.46 (CH 3 COO); 16.20/16.08 J 6 Hz.

( 2- ethoxy- 2- oxo- l, 2- oxaphosphorinan- 5- yl) methanol ( racemic cis- trans- mixture

VSC 648 (1.05 g; 3.24 mmol) was dissolved in 16 mL of a 0.5 molar solution of sodium ethoxide in ethanol. The solution was heated to 50°C and then held at 37°C for 2 hours. After evaporation to dryness in vacuo, the residue was extracted with ethyl acetate and purified by flash chromatography on silica with ethyl acetate + ethanol (9+1) as eluent. Yield 0.462 g (74%) VSC 650 in an isomer ratio (cis-trans) of 0.36/1.00.

TLC Rf 0.26 (SiO 2 ; ethyl acetate + ethanol 9+1).

<13>C NMR (CDCl3, TMS, 50 MHz); major isomer - minor isomer):

S 72.22/72.08 - 70.64/70.52 (CH 2 O in ring, J 6 Hz); 62.18 - 63.64 (CH 2 OH); 60.81/60.69 - 61.35/61.23 (CH 2 -O-P, J 6 Hz); 38.87/38.75 - 37.39/37.27 (CH, J 6 Hz); 24.69/24.52 - 23.35/23.18 (CH 2 -P, J 8 Hz); 23.06/20.48 - 21.38/18.80 (CH 2 -C -P, J 129 Hz); 16.25/16.15 (CH3-C-O-P, J 5 Hz). 5-(bromomethyl)- 2- ethoxy- 2- oxo- l. 2- oxaphosphorinane (racemic cis- trans- mixture)

N-Bromosuccinimide (2.67 g, 15 mmol) was added portionwise to a stirred, ice-cooled solution of 2-ethoxy-2-oxo-1,2-oxaphosphorinan-5-yl)methanol (VSC 650, 1.944 g,

10 mmol) and triphenylphosphine (3.97 g, 15 mmol) in 40 mL of dichloromethane, whereupon stirring was continued for 16 h at 4°C. After evaporation in vacuo, diethyl ether (∼50 mL) was added and the mixture shaken and vigorously stirred. The crystallized triphenylphosphine oxide was removed by filtration and washed with several portions of ether. The combined extracts were evaporated to dryness and purified by flash chromatography on silica with ethyl acetate + ethanol (9+1). Yield 1.569 g (61%) of VSC 654 in a cis-trans ratio of 0.7/1.0. TLC Rf 0.57 (SiO 2 ethyl acetate + ethanol 9+1). <13>C NMR (CDCl3, TMS, 50 MHz); major isomer - minor isomers): S 71.76/71.61 (CH 2 O in ring, J 6 Hz); 60.74/60.62 - 61.20/61.08 (CH 2 -O-P, J 16 Hz); 37.56/37.44 - 37.10/36.98 (CH, J 6 Hz); 32.13 - 31.67 (CH 2 Br); 26.66/26.49 - 25.37/25.23 (CH 2 P, J 7 Hz); 22.60/20.02 - 20.90/18.32 (CH 2 -C -P, J 129.4 Hz); 16.13/16.01 (CH3-C-O-P, J 6.1 Hz). Example 7 2-amino-6-chloro-9-\(2-ethoxy-2-oxo-1.2-oxaphosphorinan-5-yl)methylIpurine and 7-isomer

A mixture of 5-(bromomethyl)-2-ethoxy-2-oxo-1,2-oxaphosphorinane (VSC 654; 0.353 g, 1.82 mmol), 2-amino-6-chloropurine (0.50 g, 2, 95 mmol) anhydrous potassium carbonate (0.50 g,

3.62 mmol), and DMF (15 mL) was stirred at room temperature for 7 days. Chloroform (30 ml) was added and after filtration the

the solution evaporated to a small volume in vacuo. The residue was purified by flash chromatography on silica (chloroform + methanol 5+1). Yield 0.282 g (45%) as a mixture of cis-trans and 7-9 isomers.

TLC Rf 0.74 and 0.68 for the 7- and 9-isomer respectively (SiO 2 , chloroform + methanol 5+1).

<1>H NMR (CDCl 3 , TMS, 200 MHz); <5 7.80 and 7.76 (s, H8, the 7- and 9-isomers); 5.4 (broad s, NH2); 4.3-4.1 (m, CH 2 0 P); 4.02 (d, CH 2 N); 2.5-2.4 and 2.1-1.7 (m, CHCH2CH2P); 1.37 (dt, CH 3 -COP). <13>C NMR (CDCl3, TMS, 50 MHz); S 159.38 (C2); 153.93 (C4); 143.50 (C8); 70.91/70.79 - 69.94/69.79 (CH 2 O in ring, J 7 Hz) 61.79 to 61.45 (2 d, CH 2 -O-P, J 7 Hz); 44.35 and 43.25 (CH 2 N); 36.68/36.56 - 35.05/34.93 (CH, J, 6 Hz); 25.88/25.74 - 24.18/24.04 (CH 2 P, J 7 Hz); 22.99/20.41 - 21.16/18.59 (CH 2 -C -P, J 129 Hz); 16.59/16.47 (CH3-C-O-P, J 6 Hz).

Biological investigations

Test I Effects of the compounds of formula I on HIV in H9 cells

Materials and methods: HIV infection of H9 cells.

H9 cells, 10<5> cells per well in a 24-well plate, suspended in 2 ml RPMI medium containing 10% fetal calf serum, 100 µg/ml penicillin, 10 µg/ml streptomycin sulfate and 2 µg/ml polybrene were exposed to HIV (HTLV-IIIB) and different concentrations of the test compounds. The plates were incubated at 37°C in 5% CO 2 for 6-7 days. The contents of each well were then homogenized with a pipette and transferred to a centrifuge tube. After centrifugation for 10 min. at 1500 rpm, the supernatant was removed and the resulting cell pellet analyzed by fixation in methanol on glass slides. Human HIV positive serum diluted 1:80 or 1:160 was added and incubated for 30 min. at 37°C. The plate was then washed with phosphate-buffered saline (PBS) containing Ca<2+> and Mg<2+>. Antihuman conjugate from sheep (FITC) was added and after a new incubation the plate was again washed with PBS. Contrast staining was done with Evans blue and after drying, the frequency of HIV antigen-containing cells was determined microscopically. The test results are shown in Table 1.

Table I shows that the test compounds are active inhibitors of HIV propagation.

Test II Cytotoxic effect

H9 cells, 2x107 cells per plate, were incubated in RPMI-1640 medium containing 10% fetal calf serum, 70 mg/l penicillin, 100 mg/l streptomycin and 10 mM hepes in or without the presence of test compounds. The number of cells per plate was determined after 48 hours. Cells incubated without test compounds then underwent two cycles of cell division.

F5000 cells, which are human embryo cells, lxl0<5> cells per plate, were incubated in Eagle's minimum medium, supplemented with Earle's salts, non-essential amino acids, 10% fetal calf serum, 10 mM hepes, 70 mg/l penicillin and 100 mg/l streptomycin in or without the presence of test compounds. The number of cells per plate was determined after 48 hours. Cells incubated without the test compounds underwent one cycle of cell division. The results are given as percent inhibition of cell proliferation when the concentration of the compound is 100 µM or 250 µM.

Table II shows that the concentrations at which the compounds exhibit toxicity far exceed the concentrations required for 50% inhibition of HIV propagation indicated in Table I.

Test III Oral bioavailability

Oral bioavailability was determined by dosing the animals (cynomologous monkeys and rats) intravenously or orally with the compounds. Blood samples were taken after appropriate intervals for determining the drug level in plasma. Appropriate pharmacokinetic calculations were then made based on plasma concentration versus time.

The table shows how the plasma concentration of VSA 671 is significantly higher after VSA 671 was given as an ester (VSC 600, VSC 641) or an ester of the 6-deoxy prodrug (VSC 610).

Claims (7)

1. Analogy method for the preparation of a therapeutically active compound of formula where R<1> is hydrogen, hydroxy, mercapto, chlorine or amino; R<2> is hydrogen, hydroxy, fluorine, chlorine or amino; R<3> and R<4>, independently of each other, are selected from hydroxy or an ester residue thereof that writes from a carboxylic acid R<6>COOH, where R<6> is hydrogen, C-j-g-alkyl or phenyl, or R<3> together with R<4> are where M is hydrogen, C 1 -C 4 alkyl or a pharmaceutically acceptable counterion; and n is 1 or 2; with the proviso that when R3 and R<4> are hydroxy, then R<1> is not hydrogen, hydroxy or chlorine, and pharmaceutically acceptable salts thereof, characterized by A. condensation of an acyclic side chain where W is a terminal leaving group, to the N-9 position of a purine derivative B. a compound of formula I where R<3> and R<4> together are is subjected to ring cleavage to form a compound wherein one of R<3> and R<4> is and the other is hydroxy. 2. Process according to claim 1 for the preparation of a compound of formula I where R<2>, R<3> and R<4> are as stated in claim 1, and R<1> is hydrogen, hydroxy, mercapto or amino, characterized by the fact that corresponding starting materials are used. 3. Process according to claim 1 or 2 for producing the compound in the form of an optical isomer, characterized by the use of corresponding starting materials or separation of the product. 4. Process according to one of claims 1-3 for the production of compounds where R<3> and R<4> are both hydroxy, characterized by the use of corresponding starting materials. 5. Method according to one of claims 1-3 for the production of compounds where R<3> is or R<3> together with R<4> are characterized by the use of corresponding starting materials. 6. Process according to one of claims 1-4 for the production of 9-(4-hydroxy-2-hydroxymethylbutyl)adenine, characterized by the use of corresponding starting materials. 7. Process according to claims 3 and 4 for the production of (R)-9-(4-hydroxy-2-hydroxymethylbutyl)adenine, characterized by the use of corresponding starting materials.