Field of the Invention

The present invention relates to the use of chemical compounds and physiologically acceptable salts thereof for the therapeutic and prophylactic control and treatment of the Acquired Immuno Deficiency Syndrome (AIDS), hepatitis B virus infections and retrovirus infections and method for such control and treatment in animal and man.

Background of the Invention

In the late seventies a new disease was reported, which subsequently was referred to as Acquired Immuno Deficiency Syndrome (AIDS). It is now generally accepted that a retrovirus referred to as Human T-cell Lymphotropic Virus (HTLV-III) or Lymphadenopathy Associated Virus (LAV) plays an essential role in the etiology of AIDS. This virus will herebelow be denoted by the new name HIV (Human Immunodeficiency Virus).

The full-blown AIDS is characterized by a profound immunodeficiency due to low numbers of a subset of lymphocyte-T-helper cells, which are one target for HIV infection. The profound immunodeficiency in the full-blown AIDS patients makes these patients highly susceptible to a variety of opportunistic infections of bacterial, fungal, protozoal or viral etiology. The etiological agents among viral opportunistic infections are often found in the herpes virus group, i.e., Herpes simplex virus (HSV), Varicella Zoster virus (VZV), Epstein-Barr virus (EBV) and, especially, cytomegalovirus (CMV). Other retroviruses affecting humans are HTLV-I and II and examples of retroviruses affecting animals are feline leukemia virus and equine infections anaemia virus.

Hepatitis B virus infections cause severe disease such as acute hepatitis, chronic hepatitis, fulminant hepatitis in a considerable number of persons. It is estimated that there are 200 million patients with chronic hepatitis B infection in the world. A considerable number

of the chronic cases progress to liver cirrosis and liver tumours. In some cases the hepatitis infections also take a rapid and severe course as in fulminant B hepatitis with about 90 % mortality. At present there is no known effective treatment against hepatitis B infections.

Prior Art

Several nucleosides of the formula I below are known compounds. Some of the compounds are described for instance in J. Med. Chem. 26(9) ,1252-7, J. Org. Chem. 43(14), 2S70-6, DD 114949, US 4247544 and GB 1601020.

Disclosure of the Invention

It has been found according to the present invention that the- compounds of the formula

wherein A is 2- ^l -deoxy-D-ribofuranosyl or /3-D-arabi nofuranosyl ;

R s hydroxy or amino;

is cycloalkyl or alkyl-substituted cycloalkyl containing 1-5 carbon atoms; saturated or unsaturated, straight or branched alkyl containing 1-5 carbon atoms which may be unsubstituted or substituted with halogen, hydroxy, mercapto, trifluoroalkyl or difluoroalkyl containing 1-3 carbon atoms, phenoxy or alkoxy containing 1-3 carbon atoms; phenyl or phenylalkyl containing 1-3 carbon atoms in the alkyl part, or a physiologically acceptable salt thereof,

present a new possibility to block the multiplication of retrovirus and hepatitis B virus, respectively, by use a nucleoside analogue of said formula. Accordingly, the nucleoside analogues of said formula and physiologically acceptable salts thereof have unobvious and beneficial properties as prophylactic and/or therapeutic agents in the control or treatment of retrovirus and hepatitis B virus infections, respectively. Said nucleosides are especially interesting as agents capable of inhibiting the activity of the HIV in man.

All retroviruses, including HIV, require an enzyme called reverse transcriptase in their natural cycle of replication.

Hepatitis B virus (HBV) is a DNA virus with a unique circular double- stranded DNA genome which is partly single-stranded. It contains a specific DNA polymerase required for viral replication. This DNA polymerase also acts as a reverse transcriptase during the replication of HBV DNA via an RNA intermediate.

The compounds of the invention can be transformed by cells or enzymes to triphosphates which inhibit the activity of reverse transcriptase of retrovirus and the activity of DNA polymerase of hepatitis B virus.

Individual compounds within the formula I may be novel.

The known compounds as well as the novel compounds of formula I are prepared in known manner as is described in the literature.

The uracil moiety of the 5-substituted deoxyuridine compounds and of the 5-substituted /3-D-arabinofuranosyluracil compounds may be converted to a cytosine moiety of the corresponding deoxycytidine and β-D-arabino- furanosylcytosine analogues, by conventional methods, the principles of which have been described for example by W.L. Sung (J. Chem, Soc. Chem. Commun. 1981, p. 1089 and J. Organic Chemistry 1982, volume 47, pages 3623-3628) and by P. Herdewijn et al . (J. Medicinal Chemistry 1985, volume 28, pages 550-555).

The following nucleoside compounds constitute part of the invention as prophylactic and therapeutic agents in control or treatment of retrovirus, especially HIV, or hepatitis B virus infections:

0

HN

1A 5-R -2'-deoxyuridine 0^-N

OH

IB 1~ # -D-arabinofuranosyl )-5-R - urac l

IC 5-R -2' -deoxycytidine

OH

ID W S-D-arabinofuranosyl )-5-R ² - cytosine

wherein R may have the following meanings

alkyl 'CHrjCHn _; ,

^■ CH--CH-CH., -C-CH- 2 i 3' i ;

CH- CH-

cycloalkyl:

^■ CH.

^■ CH CH,

^• CH,

phenyl, phenyl alkyl : — <' .

/ ^R3

-CH R ³ R ^{4 >} R ⁴

H OH

CH ₃ OH

CH ₂ CH ₃ OH

CH ₂ CH ₂ CH ₃ OH

H CH ₂ 0H

CH ₃ CH ₂ 0H

CH ₂ CH ₃ CH ₂ 0H

H SH

CH ₃ SH

CH ₂ CH ₃ SH

CH ₂ CH ₂ CH ₃ SH

H CH ₂ CH ₂ 0H

CH ₃ CH ₂ CH ₂ 0H

H 0CH ₃

H Cl

H Br

H I

CH ₃ F

CH ₃ Cl

CH ₃ Br

CH ₃ i

CH ₂ CH ₃ F

CH ₂ CH ₃ Cl

CH ₂ CH ₃ Br

^CH ₂ CH- I

-CH=CH-R

H

CH ₃

CH CH _T

^CF 3 0CH ₃

CH ₂ 0H

F

Cl

^■ C = CH-V R may be the same or different and chosen among

R ⁵ the following groups H CH

CH CHo

CF.

3 0CH ₃

CH ₂ 0H

F

Cl

^■ C≡C-R

H CH-

CHnCH

^CF 3 0CH ₃

CH ₂ 0H

F

Cl

-CH=C=CH

2

H

CH ₃

CF ₃

0CH ₃

CH ₂ 0H

F

Cl

Nucleosides containing uracil as base are preferred, as agents for use in control or treatment of retrovirus, especially HIV and hepatitis B virus infections in animal and man. The following nucleosides are the most preferred compounds: 5-cyclopropyl-2'-deoxyuridine, 5-ethyl -2 ' -deoxyuri di ne , 5-hydroxymethyl -2 ' -deoxyuri di ne , 5-(l-propenyl ) -2' -deoxyuri dine, l-(β-D-arabinofuranosyl )-5-(l-propenyl )- uracil, 5-isopropeny 1-2' -deoxyuri dine, or a physiologically acceptable salt thereof.

In clinical practice the nucleosides of the 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 optionally in the form of a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier which may be a solid, semi -sol id or liquid diluent or an ingestible capsule. The compound may also be used without carrier material. As examples of pharmaceutical preparations may be mentioned tablets, dragees, capsules, granulates, suspensions, elixirs, syrups, solutions etc. Usually the active substance will comprise between 0.05 and 20 % for preparations intended for injection and between 10 and 90 % for preparations intended for oral admin stration.

In the treatment of patients suffering from retrovirus, especially HIV, or hepatitis B virus infections, it will be preferred to administer the compounds orally, parenterally or intravenously. The dosage at which the

active ingredients are administered may vary within a wide range and will depend on various factors such as the severity of the infection, the age of patient etc., and may have to be individually adjusted. As a possible range for the amount of the compounds of the invention or a physiologically acceptable salt thereof to be administered per day may be mentioned from about 10 mg to about 10 000 mg, preferentially 100-500 mg for intravenous administration and preferentially 100-1000 mg for oral administration.

Salts

The physiologically acceptable salts are suitable acid addition salts, preferably derived from non-toxic acids. Such acid addition salts include, for example, those derived from hydrochloric acid, hydroiodic acid, sulphuric acid, p-toluenesulphonic acid, methane sulphonic acid, maleic acid, lactic acid, citric acid, tartaric acid,- succinic acid, oxalic acid, p-chlorobenzenesulphonic acid, phosphoric acid, acetic acid, gluconic acid, pantothenic acid and lactobionic acid.

Experimental test

Test I. Effect of compounds of the formula I of HIV in H9 cells

Materials and methods: HIV infection of H9 cells

5 H9 cells, 10 cells per well on a 24 well plate, suspended in 2 ml

RPMI-mediu containing 10% fetal calf serum, 100 μg/ml penicillin, 10 μg/ml streptomycin sulfate and 2 μg/ml polybrene are exposed to HIV (HTLV-IIIg) and different concentrations of the inhibiting compounds. The plates are incubated at 37°C in 5% CO- for 6-7 days. The contents in each well is then homogenized with a pipette and transferred to a centrifuge tube. After centrifugation for 10 min at 1500 rpm the supernatent is removed and the cell pellet is analyzed by fixing in methanol on glass plates. Human HIV positive serum diluted 1:80 or 1:160 is added and incubated for 30 min at 37°. The plate is then washed with

2+ 2+ phosphate-buffered saline (PBS) containing Ca and Mg . Sheep antihuman conjugate (FITC) is added and after a new incubation the plate

is again washed with PBS. Contrast staining is done with Evans blue and after drying the frequency of HIV antigen containing cells is determined in a microscope. The test result is shown in Table 1.

Table 1. Concentration (μM) for 50% inhibition (IC _™ ) of human immuno- deficiency virus multiplication in cell culture

Compounds IC 5 _cn 0 (μM)

5-cyclopropy -2' -deoxyuri dine -.!

5-ethyl-2'-deoxyuridine 1

5-hydroxymethyl-2'-deoxyuridine < 1 5- ( 1 -propenyl ) -2 ' -deoxyuri di ne < 1

1-( -D-arabinofuranosyl ) -5- (1 -propenyl )-uracil <1