WO2000054771A1 - The use of 3,5-pyrazolidinedione derivatives to combat viral infections - Google Patents

The use of 3,5-pyrazolidinedione derivatives to combat viral infections Download PDF

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WO2000054771A1
WO2000054771A1 PCT/NO2000/000086 NO0000086W WO0054771A1 WO 2000054771 A1 WO2000054771 A1 WO 2000054771A1 NO 0000086 W NO0000086 W NO 0000086W WO 0054771 A1 WO0054771 A1 WO 0054771A1
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alkyl
compound according
groups
substituted
phenyl
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PCT/NO2000/000086
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French (fr)
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Toril Garatun Krogdal
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Normedica As
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • TECHNICAL FIELD This invention relates to specified substances and compositions thereof that reduce viral replication by interacting with infected cells including macrophages, monocytes and T cells from vertebrates, including mammals, particularly humans, protecting them from developing viral infection.
  • This inhibition constitutes a therapy for viral diseases including HIV/AIDS and measles.
  • this invention relates to the new action of such compounds and compositions comprising such compounds and related methods of treatment for viral diseases.
  • this invention pertains to methods for treating HIV/AIDS.
  • this invention pertains to the treatment of measles and other viral diseases (Goetz, 0. And Peller, P., Klin. achr. 50, 751- 753 (1972) ) .
  • the definition of the class to be protected is the inclusion of the necessary submolecular group and derivatives thereof in any given member of the class to produce this inhibition.
  • HIV Human immune deficiency virus
  • monocytes and T-cells.
  • AIDS Acquired immune deficiency syndrome
  • AIDS is characterized by extensive immunosuppression that predisposes patients to life-threatening opportunistic infections as well as unusual forms of neoplasm.
  • Type I HTLV-I
  • HTLV-II Type I
  • the role of HTLV-II in pathogenesis remains unclear, although it has been associated with rare cases of the T-cell variant of hairy cell leukemia (Golde et al. (1986), Seminars in Hematol. 23:3-9) .
  • the present invention contemplates the use of a class of compounds, and compositions thereof, for treating viral diseases including HIV/AIDS and measles by reducing metabolic activity in cells infected with HIV/AIDS, measles and other viral diseases thereby reducing viral production and potentially killing the infected cell.
  • the present invention also pertains to methods for treating viral infections, including HIV/AIDS and measles as well as combination therapies.
  • R 1 and R 2 which are similar or different, are chosen from the group comprising hydrogen; alkyl; functionalised alkyl; phenyl; benzoyl which may be substituted in any position with one or more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2, 3 or 4 and which may be substituted in any position, with the exception of position 1, with one or more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; 2-thiazolyl which may be substituted in the 4 and 5 positions with one or two of the groups alkyl and phenyl;
  • R 3 and R 4 being similar or different, are chosen from the group comprising hydrogen; hydroxy; halogen; alkyl; functionalised alkyl; hydroxyalkyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenylamino and diphenylamino wherein the phenyl groups may be similar or different; aminoalkyl, (alkylamino) alkyl, (dialkylamino) alkyl,
  • compositions comprising them for inhibiting cellular functions to combate viral production.
  • Ci- ⁇ alkyl as used herein, means a C ⁇ _ 6 alkyl which may be straight, branched or cyclic, optionally substituted with phenyl.
  • functionalised C ⁇ _ 6 alkyl means a C ⁇ - 6 alkyl which may be completely or partially substituted with deuterium, halogen, phenyl, alkanoyl, hydroxy, nitro, 1, 3-dioxolan-2-yl, alkylthio, phenylthio, phenylsulphinyl and phenylsulphonyl, 2-oxiranyl, 3-alkyloxiranyl and 3,3- dialkyloxiranyl, 2-furfuryl and 4-oxo-4H-l-benzopyran-3-yl .
  • phenyl as used herein, means phenyl and phenyl which may be completely or partially deuterated in any position, or substituted in one or more positions by one or more of the groups alkyl, alkoxy including 2-methoxy- ethoxymethoxy, phenoxy, alkanoyl, acyloxy, halogen, hydroxy, nitro and nitroso;
  • hydroxyalkyl means C ⁇ . - 6 alkyl as defined supra and wherein one or more hydroxy groups are attached to any position on the alkyl.
  • alkanoyl as used herein, means C ⁇ _ 6 alkanoyl wherein the alkyl group attached to the carbonyl group is substituted with hydrogen, alkyl, alkylamino, dialkylamino, phenylamino and diphenylamino, or with pyrrolidinyl and 2, 6-dimethyl-l-piperidinyl;
  • oxoalkyl as used herein, means C ⁇ - 6 oxoalkyl wherein alkyl is as defined supera.
  • alkenyl as used herein, means C 2 _6alkenyl which may be straight, branched or cyclic and optionally substituted with one or more of the groups amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different.
  • alkoxy 7 ' as used herein, means C ⁇ _ 6 alkoxy wherein the alkyl group being attached to the oxygen is as defined supra .
  • acyloxy as used herein, means C ⁇ _ 6 acyloxy wherein the alkanoyl group being attached to the oxygen is as defined supra.
  • terapéutica salt also comprises the solvates that the compounds of formula (I) as well as the salts thereof, are able to form.
  • Such solvates are for example hydrates, alcholates an the like.
  • More preferred compounds having formula (I) or therapeutic acceptable salts thereof are compounds wherein:
  • R 1 and R 2 which are similar of different, are chosen from the group comprising hydrogen; alkyl; functionalised alkyl; phenyl; benzoyl which may be substituted in any position with one or more of the groups alkoxy, alkyl, fluorine, chlorine, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2, 3 or 4 and which may be substituted in position
  • 2-thiazolyl which may be substituted in the 4 and/or 5 positions with one or two of the groups alkyl and phenyl;
  • R 3 and R 4 being similar or different, are chosen from the group comprising hydrogen; hydroxy; fluorine; alkyl; functionalised alkyl; hydroxyalkyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenylamino and diphenylamino wherein the phenyl groups may be similar or different; aminoalkyl, (alkylamino) alkyl, (dialkylamino) alkyl,
  • (4-methylmorpholinyl) alkyl (4-methylmorpholinyl) alkyl; benzoyl which may be substituted in any position with one ore more of the groups alkoxy, alkyl, fluorine, chlorine, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2,3 or 4 and which may be substituted in position
  • Oxyphenbutazone i.e., 4-butyl-l- (4- hydroxyphenyl) -2-phenyl-3, 5-pyrazolidinedione, having formula (II)
  • Figure 2 shows the cytotoxic effect of AZT after 4 days of incubation.
  • Figure 3 shows the cytotoxic effect of Oxyphenbutazone (oxyPB) .
  • Figure 4 shows the cytotoxic effect of oxyPB after 4 days of incubation.
  • Figure 5 shows the effect of AZT on viral replication.
  • Figure 6 shows the effext of oxyPB on viral replication.
  • Figure 7 shows the effect of OxyPB on Taq Polymerase.
  • the aims of this study were to evaluate the cytotoxic effects and the antiviral effects with respect to inhibition of HIV growth in vi tro in tissue cultures.
  • the effect of the compounds on cell growth and virus production was compared with AZT.
  • Sup Tl is derived from a Non- Hodgkin's T-cell lymphoma patient (Smith SD et al (1984), Cancer Research, 44, 5657) and was a gift from Dr. J. Sodroski at the Division of Human Retroviruses, Dana Farber Cancer Institute, Harvard Medical School, Boston, US.
  • the Jurkat cell line has a similar origin and was obtained from the American Tissue Culture Collection (ATCC) .
  • ATCC American Tissue Culture Collection
  • the cells were cultivated as suspensjon cultures in plastic flasks ( ⁇ unc) in RPMI 1640 medium, (Bio Whittaker) supplemented with 5% Foetal Calf Serum, 2 mM glutamine (both from Bio Whittaker) and ABAM (Anti Biotic Anti Mycotic) in 1 mM final concentrations (Sigma Chem. Company) .
  • the Anti Biotic Anti Mycotic solution consists of penicillin and fungizone. Gentamicine (Bio Whittaker) is added to a final concentration of 50 ⁇ g/ml.
  • Counting of cell numbers was performed the same day the experiments started using the Trypan blue exclusion method and a Burker counting chamber at a magnification of 400X.
  • the ratio between live and dead cells was at least 95/5 in all experiments and prior to the experiments the medium was half-changed.
  • the cell density was adjusted to approximately lxlO 6 cells/ml and kept at this concentration throughout the experiments.
  • the MTT assay method for determining the number of viable cells is the MTT assay method for determining the number of viable cells .
  • Standard curve for the MTT assay is established by diluting exponentially growing SupTI cells at known cell numbers in standard medium into 96 wells tissue culture plates ( ⁇ unc) at a total volume of 100 ⁇ l followed by adding 50 ⁇ l of MTT reagent (3 mg/ml in PBS) to each well. After addition of the MTT reagent, the plates are incubated at 37°C and 5% C0 2 for 3 hours. Then the cells are centrifuged at 2000 rpm (800x g) for 10 minutes in a centrifuge equipped with micro-titer plate holders. After centrifugation 100 ⁇ l supernatant is removed from the wells. For this purpose a multi-channel micro-pipette is used.
  • pelleted cells are resuspended in 100 ⁇ l DMSO and the plates are shaken slowly for about 10 min at room temperature before the absorption is read in a Titertek
  • Multiscan Plus MK II (ELISA reader) photometer equipped with a 580 nm light filter. A near linear relationship between the amount of cells and the intensity of staining was obtained.
  • the cells were maintained in 96 wells micro-titer plate. To each well lxlO 4 cells in 100 ⁇ l medium were added. To the suspension of cells was then added 10 ⁇ l of the test substance in water and as diluent RPMI 1640 medium was used. At the end of incubation with the compounds, survival of cells was measured by adding the MTT reagent and the samples were processed as described above.
  • the initial screening of anti-viral effects of the test compounds was based on measuring the formation of syncytia in relation to concentration of the compounds. For this purpose, cells were grown in micro-titer plates for fast screening and in T25 flasks ( ⁇ unc) for rriore accurate measurements .
  • Micro-titer plates are used to screen a large number of test-substances using lxlO 4 cells/well. It is previously experienced that this technique is suitable when screening the test-substances, but exact number of syncytia is difficult to obtain due to aggregation of cells in the small wells.
  • HIV-1 containing supernatant from Molt 3 IIIB cell-supernatant is prepared by centrifuging the cells at 1000 rpm for 5 mill.
  • the Molt 3 IIIB cell line is producing HIV virus particles constitutively.
  • the cell line was established in our laboratory by infecting Molt 3 cells (American Type Culture Collections, ATCC CRL 1552,) with the HIV-1 strain HTLV IIIB obtained from Dr. W.A. Haseltine at the Division of Human Retroviruses, Dana Farber Cancer Institute, Harvard Medical School, Boston, US.
  • Each virus supernatant to be used in the experiment should have a p24 Ag antigen concentration of 1.5 -2 ng/10 5 cells.
  • Each flask was filled with lxlO 6 cells/ml in a total volume of 5 ml. The test substances were added 30 minutes prior to the addition of the virus containing supernatant and during this preincubation time the flasks were kept at 37°C and 5% C0 2 .
  • Oxyphenbutazone is a better anti-viral agent than AZT.
  • the data presented show (indicate) that different cell types are sensitive to the specified substances at different concentrations of the drugs. An antiviral effect will therefore be explained by the fact that infected cells of the macrophage, monocyte or T-cell lineages are sensitised following infection and production of HIV and other cell types will be sensitised by other viruses infecting them.
  • Taq Polymerase is a thermostable DNA polymerase isolated from Thermus Aqua ticus . Taq Polymerase is stable at temperatures up to 95°C and displays the highest activity at 72°C.
  • the PCR involves a repeated series of temperature variations corresponding to denaturation of the template, annealing of the primers and the final extension step.
  • the effect of the high and rapid variations in temperature on the compound is not known. This must be taken into consideration when judging the results.
  • the pSVC21 plasmid containing the full length HIV-1 genome (HxB2) was used as a template and primers spanning the RT encoding region in the Pol gene were used to amplify the region encoding HIV-1 RT.
  • the amplified region corresponds to a 1681 bp DNA fragment easily detected on an agarose gel .
  • a 24-sample reaction-mixture was prepared (12 ⁇ l lOx PCR buffer, 72 ⁇ l RT5 N 72 ⁇ l RT3 N 120 ⁇ l dNTP, 24 ⁇ l Taq Polymerase, 168 ⁇ l ddH 2 0, 24 ⁇ l pSVC21) . Twenty-five ⁇ l of this mixture was transferred to a PCR tube already containing the 25 ⁇ l of the compound. The reaction mixture was briefly heated to room temperature when added to the compound to avoid precipitation.
  • the reaction was run on a Perkin Elmer 2400 thermocycler .
  • the PBS used for dissolving the compound, inhibited the PCR almost completely and this necessitated a new method for dissolving the compound.
  • a 100 mM concentration of the compound was prepared in 100% ethanol. It was attempted to further dilute the compound down to 5 mM ddH 2 0. The compound did precipitate strongly at this concentration.
  • the compound dissolves readily in PBS at this concentration At 1 mM the compound seemed to be soluble.
  • the concentration of EtOH at this concentration of the compound is 1%.
  • Amplification of HIV-1 RT was purely coincidental and has nothing to do with the proposed application of the compound.
  • Figure 7 shows the product of PCR analysed on an agarose gel wherein two parallels of the reactions were run.
  • the numbers refer to both the upper and lower wells of the gel.
  • the size of the band (in bp) corresponds to what is being expected.
  • the digits refer to
  • a tablet including the active substance according to the present invention, Oxyphenbutazone may be formulated as follows:
  • Antiviral drugs may have several ways of interacting with the recipient cells, e.g. by blocking the entry of the virus into the cell (blocking receptors, camouflaging the cell from the virus etc), by interfering with the virus' replication on an intra-cellular level (inhibiting enzymes crucial for viral replication, inhibiting integration or autonomal replication of viral D ⁇ A inside the cell, inhibiting the assembly of viral progeny etc.) or by interfering with the liberation of viruses from the infected cells. It will also be possible to combat viral infection by making the infected cells more susceptible to drugs killing them or making such cells suicidal.
  • the invention further relates to the use of the compounds having formula (I), and especially Oxyphenbutazone, according to the invention together with pharmacological adjuvants and vehicles that are physiologically compatible and not detrimental for the action of the compounds in pharmaceutical compositions.
  • pharmacological adjuvants and vehicles that are physiologically compatible and not detrimental for the action of the compounds in pharmaceutical compositions.
  • Such substances are described in Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980) .
  • the suitable dose differs and depends, for example, on the host, the type of administration, and the type and severity of the conditions to be treated, but in general, satisfactory results are to be expected in vertebrate with daily doses of 0.1 to 3000 mg/kg of body weight.
  • daily doses 0.1 to 3000 mg/kg of body weight.
  • large mammals e.g., humans
  • Preferred are values of 1 to 500 mg per day, and most preferred is 2 to 30 mg per day.
  • 200 mg of Oxyphenbutazone is equal to 15 ⁇ mol of the compound.
  • the daily dose of the compositions including the compounds according to the invention should be administered in 1 to 3 partial doses per day.
  • the compounds according to the invention can be administered by any usual method in the case of systemic treatment, especially enterally, preferably parenterally, such as subcutaneously, intravenously, intradermally and orally, and most preferably orally. Tablets, capsules, drops, suppositories, injection solutions, or suspensions are the appropriate forms for administration.
  • Combination therapies are also contemplated by the invention wherein a compound according to the invention having formula (I) or (II) is used for preparing a composition including additionally an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor for treating viral infections, especially HIV/AIDS and measles.
  • the invention further relates to a method of treating a viral infection, especially HIV/AIDS and measles, wherein a compound according to the invention having formula (I) or (II) is administered to a subject in need of such a treatment .
  • a method of treating a viral infection comprises to administer a compound or pharmaceutical composition according to the present invention systemically, especially enterally, preferably parenterally, such as subcutaneously, intravenously, intradermally and orally, and most preferably orally.
  • a method of treating a viral infection does also include to administer a compound according to the invention together with an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor for treating viral infections, especially HIV/AIDS and measles.

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Abstract

The invention relates to the use of a compound having formula (I), wherein R?1, R2, R3 and R4¿ are as specified in the specification, preferably the compound is Oxyphenbutazone, and pharmaceutical salts thereof for preparing a pharmaceutical composition for inhibiting viral infections, especially HIV/AIDS and measles. Moreover, the invention concerns related methods of treating the same.

Description

THE USE OF 3, 5-PYRAZOLIDINEDIONE DERIVATIVES TO COMBAT
VIRAL INFECTIONS
TECHNICAL FIELD This invention relates to specified substances and compositions thereof that reduce viral replication by interacting with infected cells including macrophages, monocytes and T cells from vertebrates, including mammals, particularly humans, protecting them from developing viral infection. This inhibition constitutes a therapy for viral diseases including HIV/AIDS and measles. More particularly, this invention relates to the new action of such compounds and compositions comprising such compounds and related methods of treatment for viral diseases. In one specific aspect, this invention pertains to methods for treating HIV/AIDS. In a second specific aspect, this invention pertains to the treatment of measles and other viral diseases (Goetz, 0. And Peller, P., Klin. achr. 50, 751- 753 (1972) ) . The definition of the class to be protected is the inclusion of the necessary submolecular group and derivatives thereof in any given member of the class to produce this inhibition.
BACKGROUND OF THE INVENTION Development of antiviral drugs with direct viral targets (RT and protease inhibitors, integrase inhibitors under development) has so far resulted only in drugs severely exposed to resistance development (Pathogenesis of HIV/AIDS) . As a result drugs with general cytotoxic mechanisms have been tested for specificity against viral infections (e.g., Thalidomide) . The benefit of a reasonably specific cytotoxic substance is the lack of exposure to resistance development. To replicate and produce progeny virus all viruses are strongly dependent on the metabolism and metabolizing machinery of the cells they infect. It is also well documented that all viruses interfere with the metabolism of the infected cell thereby modifying the cell in a number of different ways. For some virus/cell systems the ultimate outcome of an infection is cell death, for other systems cell transformations are observed and for other systems only minor changes of the infected cell can be detected.
Human immune deficiency virus (HIV) infects human cells including macrophages, monocytes and T-cells. Acquired immune deficiency syndrome (AIDS) results from infection with HIV.
AIDS is characterized by extensive immunosuppression that predisposes patients to life-threatening opportunistic infections as well as unusual forms of neoplasm. As to the other known subgroups or types of human T-lymphotropic viruses, Type I (HTLV-I) is believed to cause T-cell proliferation in leukemia. The role of HTLV-II in pathogenesis remains unclear, although it has been associated with rare cases of the T-cell variant of hairy cell leukemia (Golde et al. (1986), Seminars in Hematol. 23:3-9) .
Synthesis of DΝA complementary to viral RΝA is required for both retroviral integration into host DΝA and for the generation of new virions. For this reason, the HIV-encoded reverse transcriptase is a logical target for the development of agents for the treatment of patients with the acquired immunodeficiency syndrome (De Clercq et al. (1986) J. Med. Chem., 29:1561-1569), and with other diseases of retroviral origin. Mitsuya et al. ((1985) Proc. Νatl. Acad. Sci. USA, 82:7006- 7100) reported that 3 ' -azido-3 ' -deoxythymidine (AZT) blocked the replication of HIV in cultured human T lymphoblasts, and inhibited the cytopathic effects of the virus. AZT was presumably phosphorylated by the T-cells and converted to the 5' -triphosphate derivative. That derivative was reported by those authors to be an inhibitor of HIV reverse transcriptase activity. Yarchoan et al. ((1986) Lancet, i:575-580), administered AZT to patients with AIDS or AIDS-related disease complexes. The drug was reportedly well tolerated and crossed the blood/brain barrier.
Observations made in clinical trials with AZT have shown that treatment of patients with AIDS or AIDS-related complex with AZT has resulted in elevation of CD4 (T4) peripheral blood cell counts, restoration of cutaneous delayed hypersensitivity, and reduction of the rate of opportunistic infections and death; results that can be related to the effect of AZT on T-cells.
From EP 0 237 796 A3 it has previously been suggested to use a prostaglandin synthesis inhibitor for manufacturing an anti-AIDS medicament, the relevance of this document to the present invention being that the compound oxyphenbutazone is mentioned among several others to be used in this manner. However, no actual data relating to the effect of oxyphenbutazone is given therein. This information concerning oxyphenbutazone has then to be considered on the basis of a subsequent patent application US Patent 4.956.377.
The observations referred in US Patent 4,956,377 provided an indication that a therapeutic effect against HIV might be possible. The patent describes a specific substance and specific mechanics, both of which were abandoned after conducting formal studies leading the person skilled in the art to presume that this was a non-viable line of pharmaceuticals for viral diseases.
The present discovery that this class of substances will reduce replication of infected cells can be used to inhibit viral disease in vertebrates, including mammals, particularly human HIV/AIDS and measles, and has proven to possess a better inhibitory effect than AZT.
SUMMARY OF THE INVENTION
The present invention contemplates the use of a class of compounds, and compositions thereof, for treating viral diseases including HIV/AIDS and measles by reducing metabolic activity in cells infected with HIV/AIDS, measles and other viral diseases thereby reducing viral production and potentially killing the infected cell. The present invention also pertains to methods for treating viral infections, including HIV/AIDS and measles as well as combination therapies.
DETAILED DESCRIPTION OF THE INVENTION The present invention concerns the use of compounds having formula (I) :
Figure imgf000007_0001
or therapeutic acceptable salts thereof,
wherein
R1 and R2, which are similar or different, are chosen from the group comprising hydrogen; alkyl; functionalised alkyl; phenyl; benzoyl which may be substituted in any position with one or more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2, 3 or 4 and which may be substituted in any position, with the exception of position 1, with one or more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; 2-thiazolyl which may be substituted in the 4 and 5 positions with one or two of the groups alkyl and phenyl;
1, 2-benzisothiazol-2 (3H) -yl;
1, 2-benzisothiazol-2 (3H) -yl S, S-dioxide;
3-oxo-l, 2-benzisothiazol-2 (3H) -yl S,S dioxide; 1H-1, 2, 4-triazol-3-yl which may be substituted in position
5 with one of the groups alkyl and phenyl;
R3 and R4, being similar or different, are chosen from the group comprising hydrogen; hydroxy; halogen; alkyl; functionalised alkyl; hydroxyalkyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenylamino and diphenylamino wherein the phenyl groups may be similar or different; aminoalkyl, (alkylamino) alkyl, (dialkylamino) alkyl,
(phenylamino) alkyl and (diphenylamino) alkyl wherein the alkyl groups may be similar or different, and wherein the phenyl groups may be similar or different; (4-methylmorpholinyl) alkyl; benzoyl which may be substituted in any position with one ore more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2,3 or 4 and which may be substituted in any position, except for position 1, by one or more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; alkanoyl; oxoalkyl which may be substituted in any position with alkyl or phenyl; phenylazo; alkenyl which may be completely or partially substituted with deuterium, halogen, nitro, phenylthio, phenylsulphinyl and phenylsulphonyl; 2, 3-dihydro-l, 3-dioxo-lH-indene-2-yl which may be substituted in one or more positions with one or more of the groups alkyl, acyloxy, hydroxy and halogen;
1, 3-dihydro-3-oxo-l-isobenzofuranyl; phenyl (phenylimino) methyl which may be completely or partially deutetrated in any position, or substituted in one or more positions with one or more of the groups alkoxy, alkanoyl, alkyl, halogen, hydroxy and nitro; phenyl; 2-furfuryl; lH-indol-3-yl;
(lH-indol-3-yl) methyl;
and wherein R3, R4 is the methylidene group =C (R5) R6 wherein R5 and R6 are similar or different and are chosen form the group comprising hydrogen; hydroxy; alkyl which may be completely or partially substituted with halogen, preferably fluorine and chlorine; alkenyl; phenyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenymethyleneamin; 2-furanyl; lH-indol-2-yl; lH-indol-3-yl;
or are 2, 5-bis [ (3-ethyl-2 (3H) -benzothiazolylidene) ethylidene] - cyclopentyliden; cycloalkylidene with 4 or more C-atoms in the ring; diazo;
4- [ (4-chlorophenyl)methylene] -5-thioxo-3-isothiazol- idinylidene;
1, 5-dihydro-5-oxo-2H-pyrrol-2-ylidene which may be substituted in position 1 with one of the groups phenyl and/or in position 5 with the groups phenylhydrazono or phenylimino; dihydro-5-oxo-2 (3H) -furanylidene : 3, 5-dioxo-4-pyrazolidinylidene;
4 (1H) -quinolinylidene;
2- (3-ethyl-2 (3H) -benzothiazolylidene-1- [ (3-et yl-2 (3H) - benzothiazolylidene) methyl] ethylidene;
2- (3-ethyl-2 (3H) -benzothiazolylidene-1- [ (l-ethyl-3, 4- dihydro-2 (1H) -quinolinylidene) methyl] ethylidene;
2- (3-ethyl-2 (3H) -benzothiazolylidene-1- [ (l-ethyl-2 (1H) - quinolinylidene) methyl] ethylidene;
(3-ethyl-2-oxazolidinylidene) ethylidene,
as well as compositions comprising them, for inhibiting cellular functions to combate viral production.
The term "Ci-βalkyl" as used herein, means a Cι_6alkyl which may be straight, branched or cyclic, optionally substituted with phenyl.
The term " functionalised Cι_6alkyl" as used herein, means a Cι-6alkyl which may be completely or partially substituted with deuterium, halogen, phenyl, alkanoyl, hydroxy, nitro, 1, 3-dioxolan-2-yl, alkylthio, phenylthio, phenylsulphinyl and phenylsulphonyl, 2-oxiranyl, 3-alkyloxiranyl and 3,3- dialkyloxiranyl, 2-furfuryl and 4-oxo-4H-l-benzopyran-3-yl .
The term "phenyl" as used herein, means phenyl and phenyl which may be completely or partially deuterated in any position, or substituted in one or more positions by one or more of the groups alkyl, alkoxy including 2-methoxy- ethoxymethoxy, phenoxy, alkanoyl, acyloxy, halogen, hydroxy, nitro and nitroso;
The term "hydroxyalkyl" as used herein, means Cτ.-6alkyl as defined supra and wherein one or more hydroxy groups are attached to any position on the alkyl. The term "alkanoyl" as used herein, means Cι_6alkanoyl wherein the alkyl group attached to the carbonyl group is substituted with hydrogen, alkyl, alkylamino, dialkylamino, phenylamino and diphenylamino, or with pyrrolidinyl and 2, 6-dimethyl-l-piperidinyl;
The term "oxoalkyl" as used herein, means Cι-6oxoalkyl wherein alkyl is as defined supera.
The term "alkenyl" as used herein, means C2_6alkenyl which may be straight, branched or cyclic and optionally substituted with one or more of the groups amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different.
The term "alkoxy7' as used herein, means Cι_6alkoxy wherein the alkyl group being attached to the oxygen is as defined supra .
The term "acyloxy" as used herein, means Cι_6acyloxy wherein the alkanoyl group being attached to the oxygen is as defined supra.
The term "therapeutic salt" as used herein, also comprises the solvates that the compounds of formula (I) as well as the salts thereof, are able to form. Such solvates are for example hydrates, alcholates an the like.
More preferred compounds having formula (I) or therapeutic acceptable salts thereof are compounds wherein:
R1 and R2, which are similar of different, are chosen from the group comprising hydrogen; alkyl; functionalised alkyl; phenyl; benzoyl which may be substituted in any position with one or more of the groups alkoxy, alkyl, fluorine, chlorine, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2, 3 or 4 and which may be substituted in position
5 with one of the groups fluorine and nitro;
2-thiazolyl which may be substituted in the 4 and/or 5 positions with one or two of the groups alkyl and phenyl;
R3 and R4, being similar or different, are chosen from the group comprising hydrogen; hydroxy; fluorine; alkyl; functionalised alkyl; hydroxyalkyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenylamino and diphenylamino wherein the phenyl groups may be similar or different; aminoalkyl, (alkylamino) alkyl, (dialkylamino) alkyl,
(phenylamino) alkyl and (diphenylamino) alkyl wherein the alkyl groups may be similar or different, and the phenyl groups may be similar or different;
(4-methylmorpholinyl) alkyl; benzoyl which may be substituted in any position with one ore more of the groups alkoxy, alkyl, fluorine, chlorine, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2,3 or 4 and which may be substituted in position
5 with fluorine, chlorine, hydroxy or nitro; alkanoyl; oxoalkyl which may be substituted in any position with alkyl or phenyl; phenylazo; alkenyl; phenyl (phenylimino) methyl; phenyl;
2-furfuryl; lH-indol-3-yl;
(lH-indol-3-yl) methyl;
and wherein R , R is the methylidene group =C(R5)R6 wherein R5 and R6 being similar or different, are chosen form the group comprising hydrogen; hydroxy; alkyl which may be completely or partially substituted with fluorine and chlorine; alkenyl which may be substituted with one or more phenyl groups, and which alkenyl may be completely or partially substituted with deuterium, fluorine, chlorine, nitro, phenylthio, phenylsulphinyl and phenylsulphonyl; phenyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; pheny ethyleneamin;
2-furanyl; lH-indol-2-yl; lH-indol-3-yl.
The compound named Oxyphenbutazone, i.e., 4-butyl-l- (4- hydroxyphenyl) -2-phenyl-3, 5-pyrazolidinedione, having formula (II)
Figure imgf000014_0001
represents an especially preferred embodiment of the present invention.
It is to be understood that the successful application of derivatives within the indicated group of these compounds are used to inhibit viral diseases as described herein was quite unexpected.
It has been known for some time that ΝSAIDs (non-steroid anti-inflammatory drugs) , particularly antiphlogistic compounds, previously used to treat rheumatoid disorders might have an effect on viral disease (Steinmeyer&Kalbhen, Inflam . Res. 1996 Jul; 45 (7 ) : 324-9) . However, due to the great number of compounds in this group and lack of understanding of the underlying mechanism it has prior to the present invention not been possible to identify any specific compound with sufficient therapeutic effect.
Νon-steroid anti-inflammatory compounds used against rheumatoid disorders were previously thought to inhibit the synthesis of prostagladines . However, their method of action is still unclear.
The results of the laboratory studies described in detail below show that Oxyphenbutazone reduces the HIV virus count more than AZT, a well known therapy for HIV/AIDS. The results presented are not intended to limit the scope of the invention in any way.
DESCRIPTION OF THE DRAWINGS Figure 1 shows the cytotoxic effects of AZT.
Figure 2 shows the cytotoxic effect of AZT after 4 days of incubation.
Figure 3 shows the cytotoxic effect of Oxyphenbutazone (oxyPB) . Figure 4 shows the cytotoxic effect of oxyPB after 4 days of incubation.
Figure 5 shows the effect of AZT on viral replication. Figure 6 shows the effext of oxyPB on viral replication. Figure 7 shows the effect of OxyPB on Taq Polymerase.
EXAMPLES
Example 1
The aims of this study were to evaluate the cytotoxic effects and the antiviral effects with respect to inhibition of HIV growth in vi tro in tissue cultures. The effect of the compounds on cell growth and virus production was compared with AZT.
Cultivation of cells
In this study the two human CD4 ' lymphocyte cell lines Sup Tl and Jurkat were applied. Sup Tl is derived from a Non- Hodgkin's T-cell lymphoma patient (Smith SD et al (1984), Cancer Research, 44, 5657) and was a gift from Dr. J. Sodroski at the Division of Human Retroviruses, Dana Farber Cancer Institute, Harvard Medical School, Boston, US. The Jurkat cell line has a similar origin and was obtained from the American Tissue Culture Collection (ATCC) . These cell lines were chosen for the studies due to their high content of CD ' receptors and ability to form large syncytia following infection with HIV-1. The cells were cultivated as suspensjon cultures in plastic flasks (Νunc) in RPMI 1640 medium, (Bio Whittaker) supplemented with 5% Foetal Calf Serum, 2 mM glutamine (both from Bio Whittaker) and ABAM (Anti Biotic Anti Mycotic) in 1 mM final concentrations (Sigma Chem. Company) . The Anti Biotic Anti Mycotic solution consists of penicillin and fungizone. Gentamicine (Bio Whittaker) is added to a final concentration of 50 μg/ml.
Counting of cell numbers was performed the same day the experiments started using the Trypan blue exclusion method and a Burker counting chamber at a magnification of 400X. The ratio between live and dead cells was at least 95/5 in all experiments and prior to the experiments the medium was half-changed. The cell density was adjusted to approximately lxlO6 cells/ml and kept at this concentration throughout the experiments.
Testing of cytotoxic effects
The MTT assay method for determining the number of viable cells .
The principle of this assay is based on the cleavage of the yellow tetrazolium salt MMT (3- ( 4 , 5-dimethylthiazol-2-yl) - 2,5 diphenyltetrazoliumbromide (Thiazolyl blue) (Sigma
Chemical Company) to form purple forniazan crystals due to the dehydrogenase activity in active mitochondria present in living cells (Mosman, T et al (1983) J. Immunol. Methods, 65, 55) .
Standard curve for the MTT assay is established by diluting exponentially growing SupTI cells at known cell numbers in standard medium into 96 wells tissue culture plates (Νunc) at a total volume of 100 μl followed by adding 50 μl of MTT reagent (3 mg/ml in PBS) to each well. After addition of the MTT reagent, the plates are incubated at 37°C and 5% C02 for 3 hours. Then the cells are centrifuged at 2000 rpm (800x g) for 10 minutes in a centrifuge equipped with micro-titer plate holders. After centrifugation 100 μl supernatant is removed from the wells. For this purpose a multi-channel micro-pipette is used.
The pelleted cells are resuspended in 100 μl DMSO and the plates are shaken slowly for about 10 min at room temperature before the absorption is read in a Titertek
Multiscan Plus MK II (ELISA reader) photometer equipped with a 580 nm light filter. A near linear relationship between the amount of cells and the intensity of staining was obtained.
Effect of test substances on the growth of uninfected SupTl cells
For each substance tested 3 parallel experiments were done.
Survival of cells was tested between 2 and 7 days, respectively, after starting treatment of the cells with the substances.
The cells were maintained in 96 wells micro-titer plate. To each well lxlO4 cells in 100 μl medium were added. To the suspension of cells was then added 10 μl of the test substance in water and as diluent RPMI 1640 medium was used. At the end of incubation with the compounds, survival of cells was measured by adding the MTT reagent and the samples were processed as described above.
Results from these experiments are presented for AZT in figure 1 and for the test compound Oxyphenbutazone in figure 3 as relative cell numbers (MTT-values) as a function of dose and time (days) of incubation. In figures 2 (AZT) and 4 (Oxyphenbutazone) are the effect of the compounds on cell growth after four days of incubation shown as a function of concentration of the compounds. Almost similar curves would have been obtained using the other time points from figures 1 or 3. The main conclusion which can be drawn from these data is that both AZT (as expected from previously published reports) and Oxyphenbutazone affects the growth of these cells in culture. Furthermore it can be concluded that antiviral effects should not be studied at concentrations above lOμM since the effect on cell growth would directly influence the viral production.
The toxic doses of AZT and Oxyphenbutazone observed in these studies can not directly be related to toxic doses of these compounds in vivo since in tissue cultures the cells are directly exposed to the compounds while in vivo the picture is much more complex due to the heterogenic environment for the different cells.
If even distribution of AZT or Oxyphenbutazone is assumed in a human body of approximately 70 kg a concentration of 1 mM would have been obtained if 15 mg AZT or 18 mg Oxyphenbutazone had been given to the person.
Testing for anti-viral effects
The initial screening of anti-viral effects of the test compounds was based on measuring the formation of syncytia in relation to concentration of the compounds. For this purpose, cells were grown in micro-titer plates for fast screening and in T25 flasks (Νunc) for rriore accurate measurements .
Micro-titer plates are used to screen a large number of test-substances using lxlO4 cells/well. It is previously experienced that this technique is suitable when screening the test-substances, but exact number of syncytia is difficult to obtain due to aggregation of cells in the small wells.
To estimate the exact number of syncytia present after infection of cells by HIV-1 we have found that T25 flasks (Νunc) are more convenient. HIV-1 containing supernatant from Molt 3 IIIB cell-supernatant is prepared by centrifuging the cells at 1000 rpm for 5 mill. The Molt 3 IIIB cell line is producing HIV virus particles constitutively. The cell line was established in our laboratory by infecting Molt 3 cells (American Type Culture Collections, ATCC CRL 1552,) with the HIV-1 strain HTLV IIIB obtained from Dr. W.A. Haseltine at the Division of Human Retroviruses, Dana Farber Cancer Institute, Harvard Medical School, Boston, US.
In order to standardise the supernatant with respect to the amount of virus, p24 Ag antigen was measured using an ELISA based technique.
Each virus supernatant to be used in the experiment should have a p24 Ag antigen concentration of 1.5 -2 ng/105 cells. Each flask was filled with lxlO6 cells/ml in a total volume of 5 ml. The test substances were added 30 minutes prior to the addition of the virus containing supernatant and during this preincubation time the flasks were kept at 37°C and 5% C02.
After preincubation 500 μl of virus supernatant is added, and the number of syncytia was counted after 24 and 48 hours incubation; this being the standard times for optimal syncytia formation for these cell lines at the concentration of virus used. For each test-substance 2 flasks in parallel were used and the syncytia in each flask counted by counting the number of syncytia on 5 different places in the flask, thus giving 10 independent counts for each test-substance. The parallels obtained are usually, and should be, within 10% variation for each experiment.
The total amount of syncytia in infected cultures at different concentrations of test substances are given in tables and plotted in figures. In the graphs the results after 24 hours and 48 hours will be presented. Supernatants (0.5 ml) from the cell cultures were collected every day and were analysed with respect to HIV content measured as p24 antigen. For this purpose a commercially available ELISA was used. For the ELISA test the supernatants were centrifuged free of cells at low centrifugation forces.
Data from these studies are shown in figure 5 for AZT and in figure 6 for Oxyphenbutazone.
In figure 5 the amount of p24 antigen observed in the supernatants taken at different time points (days) are plotted against the concentration of AZT in the HIV infected cell cultures and in figure 6 the amount of p24 antigen observed in the supernatants taken at different time points (days) are plotted against the concentration of Oxyphenbutazone in the HIV infected cell cultures.
It is evident from the data presented in these two figures that both with AZT and with Oxyphenbutazone a concentration dependent reduction of virus production can be observed. From these experiments it can also be concluded that at the experimental conditions used Oxyphenbutazone is a better anti-viral agent than AZT. The data presented show (indicate) that different cell types are sensitive to the specified substances at different concentrations of the drugs. An antiviral effect will therefore be explained by the fact that infected cells of the macrophage, monocyte or T-cell lineages are sensitised following infection and production of HIV and other cell types will be sensitised by other viruses infecting them.
The reduction of HIV production at certain concentrations of the test substances described herein and experimental conditions used as well as the effect of production of viral DNA as measured by PCR reactions using the Taq- polymerase and specially designed viral primer pairs all demonstrate reduction in viral replication. The data also indicate that the viral infection and replication turns the cell more vulnerable to the specified substances.
Example 2
The Effect of Oxyphenbutazone on Taq Polymerase
Background
The effect of Oxyphenbutazone on Taq Polymerase has been investigated. Taq Polymerase is a thermostable DNA polymerase isolated from Thermus Aqua ticus . Taq Polymerase is stable at temperatures up to 95°C and displays the highest activity at 72°C.
The PCR involves a repeated series of temperature variations corresponding to denaturation of the template, annealing of the primers and the final extension step. The effect of the high and rapid variations in temperature on the compound is not known. This must be taken into consideration when judging the results.
Procedure
A previously well-established reaction was modified as to enable it to screen for inhibition of Taq Polymerase. The pSVC21 plasmid containing the full length HIV-1 genome (HxB2) was used as a template and primers spanning the RT encoding region in the Pol gene were used to amplify the region encoding HIV-1 RT. The amplified region corresponds to a 1681 bp DNA fragment easily detected on an agarose gel .
Primers RT5 5- GGGAATTCCCCATTAGCCCTATTGG-3 RT3X 5- CCGGGATCCTAGTACTTTCCTGATTC-3
The following reactions were set up:
5 μl lOx PCR buffer (Geneamp Perkin Elmer)
3 μl 15 μM RT5^ Primer
3 μl 15 μM RT3^ Primer
5 μl dNTP (2.5 mM each of dATP, dGTP, dCTP, dTTP. Promega
U1240) 1 μl Taq Polymerase (lU/μl) (Perkin Elmer AmpliTaq N808-
0155)
1 μl -0.5 ng/ μl pSVC21
7 μl ddH20
25 μl Oxyphenbutazone in 2x final concentration =50 μl total volume
A 24-sample reaction-mixture was prepared (12 μl lOx PCR buffer, 72 μl RT5 N 72 μl RT3 N 120 μl dNTP, 24 μl Taq Polymerase, 168 μl ddH20, 24 μl pSVC21) . Twenty-five μl of this mixture was transferred to a PCR tube already containing the 25 μl of the compound. The reaction mixture was briefly heated to room temperature when added to the compound to avoid precipitation.
The following program was run:
Start I 35 cycles I End 94°C I 94°C 50°C 72°C | 72°C
4 minutes 30 seconds 45 seconds 3 minutes 7 minutes
The reaction was run on a Perkin Elmer 2400 thermocycler .
The PBS, used for dissolving the compound, inhibited the PCR almost completely and this necessitated a new method for dissolving the compound. A 100 mM concentration of the compound was prepared in 100% ethanol. It was attempted to further dilute the compound down to 5 mM ddH20. The compound did precipitate strongly at this concentration.
The compound dissolves readily in PBS at this concentration At 1 mM the compound seemed to be soluble. The concentration of EtOH at this concentration of the compound is 1%.
Amplification of HIV-1 RT was purely coincidental and has nothing to do with the proposed application of the compound.
Results
There seems to be a correlation between increasing concentrations of the compound and inhibition of the PCR. The concentration of ethanol at 500 μM of the compound is 0.5%. This did not inhibit the reaction, suggesting that the compound causes the effect.
The experiments were repeated and the results were reproducible. There were some differences in the amount of inhibition, especially at lower concentrations of the compound. This is believed to be a result of the way the compound is prepared (dissolving it in water; greater instability concerning solubility of the compound) and the semi quantitative nature of the experiment.
Figure 7 shows the product of PCR analysed on an agarose gel wherein two parallels of the reactions were run. The numbers refer to both the upper and lower wells of the gel. The size of the band (in bp) corresponds to what is being expected. The digits refer to
1. lpGEM
1. Positive control (no compound X, only water)
2.5 μm X 3.10 μm X
4.20 μm X 5.50 μm X
6.100 μm X
7.150 μm X 8.200 μm X
9.300 μm X
10. 500 μm X
11. Control containing only the solvent equal to the
500 μm sample 12. Control containing only the solvent equal to the
500 μm sample
Example 3 Composition
A tablet including the active substance according to the present invention, Oxyphenbutazone, may be formulated as follows:
100 mg Oxyphenbutazone mixed with 100 mg aluminium hydroxide and 100 mg magnesium trisilicate. Treatment of virally infected animals and humans with the specific substances will lead to a reduction in viral titers and thereby improving combating the viral infection by the immune system. Furthermore, a reduction in virus production by the specified substances will reduce the production of mutated viruses giving rise to drug resistance when the patient (or host) is undergoing antiviral treatment by drugs (e.g. AZT) directly interacting with the viral replication machinery.
Antiviral drugs may have several ways of interacting with the recipient cells, e.g. by blocking the entry of the virus into the cell (blocking receptors, camouflaging the cell from the virus etc), by interfering with the virus' replication on an intra-cellular level (inhibiting enzymes crucial for viral replication, inhibiting integration or autonomal replication of viral DΝA inside the cell, inhibiting the assembly of viral progeny etc.) or by interfering with the liberation of viruses from the infected cells. It will also be possible to combat viral infection by making the infected cells more susceptible to drugs killing them or making such cells suicidal.
Initial studies of the compounds according to the present invention indicate that the compounds have an intra- cellular action interfering with the production of viruses inside the infected cells. The exact action of the compounds is at present uncertain, but its effect is at least as pronounced as AZT being a much used drug in anti- retroviral therapy.
The invention further relates to the use of the compounds having formula (I), and especially Oxyphenbutazone, according to the invention together with pharmacological adjuvants and vehicles that are physiologically compatible and not detrimental for the action of the compounds in pharmaceutical compositions. Such substances are described in Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980) .
For this therapeutic effect, the suitable dose differs and depends, for example, on the host, the type of administration, and the type and severity of the conditions to be treated, but in general, satisfactory results are to be expected in vertebrate with daily doses of 0.1 to 3000 mg/kg of body weight. With large mammals, e.g., humans, there is a recommended daily dose of 0.1 to 3000 mg of the compounds according to the invention. Preferred are values of 1 to 500 mg per day, and most preferred is 2 to 30 mg per day. 200 mg of Oxyphenbutazone is equal to 15 μmol of the compound. The daily dose of the compositions including the compounds according to the invention should be administered in 1 to 3 partial doses per day.
The compounds according to the invention can be administered by any usual method in the case of systemic treatment, especially enterally, preferably parenterally, such as subcutaneously, intravenously, intradermally and orally, and most preferably orally. Tablets, capsules, drops, suppositories, injection solutions, or suspensions are the appropriate forms for administration.
Combination therapies are also contemplated by the invention wherein a compound according to the invention having formula (I) or (II) is used for preparing a composition including additionally an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor for treating viral infections, especially HIV/AIDS and measles. The invention further relates to a method of treating a viral infection, especially HIV/AIDS and measles, wherein a compound according to the invention having formula (I) or (II) is administered to a subject in need of such a treatment .
A method of treating a viral infection comprises to administer a compound or pharmaceutical composition according to the present invention systemically, especially enterally, preferably parenterally, such as subcutaneously, intravenously, intradermally and orally, and most preferably orally.
A method of treating a viral infection does also include to administer a compound according to the invention together with an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor for treating viral infections, especially HIV/AIDS and measles.

Claims

ClaimsWha t is claimed is :
1. Use of a compound having formula (I)
Figure imgf000028_0001
or therapeutic acceptable salts thereof,
wherein
R1 and R2, which are similar or different, are chosen from the group comprising hydrogen; alkyl; functionalised alkyl; phenyl; benzoyl which may be substituted in any position with one or more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2, 3 or 4 and which may be substituted in any position, with the exception of position 1, with one or more of the groups alkoxy, alkyl, halogen, hydroxy and nitro;
2-thiazolyl which may be substituted in the 4 and 5 positions with one or two of the groups alkyl and phenyl;
1, 2-benzisothiazol-2 (3H) -yl; l,2-benzisothiazol-2 (3H)-yl S,S-dioxide;
3-oxo-l, 2-benzisothiazol-2 (3H) -yl S,S dioxide;
1H-1, 2, 4-triazol-3-yl which may be substituted in position
5 with one of the groups alkyl and phenyl;
R3 and R4, being similar or different, are chosen from the group comprising hydrogen; hydroxy; halogen; alkyl; functionalised alkyl; hydroxyalkyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenylamino and diphenylamino wherein the phenyl groups may be similar or different; aminoalkyl, (alkylamino) alkyl, (dialkylamino) alkyl,
(phenylamino) alkyl and (diphenylamino) alkyl wherein the alkyl groups may be similar or different, and wherein the phenyl groups may be similar or different;
(4-methylmorpholinyl) alkyl; benzoyl which may be substituted in any position with one ore more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2,3 or 4 and which may be substituted in any position, except for position 1, by one or more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; alkanoyl; oxoalkyl which may be substituted in any position with alkyl or phenyl; phenylazo; alkenyl which may be completely or partially substituted with deuterium, halogen, nitro, phenylthio, phenylsulphinyl and phenylsulphonyl;
2, 3-dihydro-l, 3-dioxo-lH-indene-2-yl which may be substituted in one or more positions with one or more of the groups alkyl, acyloxy, hydroxy and halogen;
1, 3-dihydro-3-oxo-l-isobenzofuranyl; phenyl (phenylimino) methyl which may be completely or partially deutetrated in any position, or substituted in one or more positions with one or more of the groups alkoxy, alkanoyl, alkyl, halogen, hydroxy and nitro; phenyl;
2-furfuryl; lH-indol-3-yl; (lH-indol-3-yl) methyl;
and wherein R3, R4 is the methylidene group =C(R5)R6 wherein R5 and R6 are similar or different and are chosen form the group comprising hydrogen; hydroxy; alkyl which may be completely or partially substituted with halogen, preferably fluorine and chlorine; alkenyl; phenyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl ; phenymethyleneamin;
2-furanyl; lH-indol-2-yl; lH-indol-3-yl;
or are
2, 5-bis [ (3-ethyl-2 (3H) -benzothiazolylidene) ethylidene] - cyclopentyliden; cycloalkylidene with 4 or more C-atoms in the ring; diazo;
4- [ (4-chlorophenyl)methylene] -5-thioxo-3-isothiazol- idinylidene; 1, 5-dihydro-5-oxo-2H-pyrrol-2-ylidene which may be substituted in position 1 with one of the groups phenyl and/or in position 5 with the groups phenylhydrazono or phenylimino; dihydro-5-oxo-2 (3H) -furanylidene : 3, 5-dioxo-4-pyrazolidinylidene;
4 (1H) -quinolinylidene;
2- (3-ethyl-2 (3H) -benzothiazolylidene-1- [ (3-ethyl-2 (3H) - benzothiazolylidene) methyl] ethylidene;
2- (3-ethyl-2 (3H) -benzothiazolylidene-1- [ (l-ethyl-3, 4- dihydro-2 (1H) -quinolinylidene) methyl] ethylidene;
2- (3-ethyl-2 (3H) -benzothiazolylidene-1- [ (l-ethyl-2 (1H) - quinolinylidene)methyl] ethylidene;
(3-ethyl-2-oxazolidinylidene) ethylidene,
for preparing a pharmaceutical composition for treating viral diseases/infections.
2. Use of a compound according to claim 1 wherein R1 and R2, which are similar of different, are chosen from the group comprising hydrogen; alkyl; functionalised alkyl; phenyl; benzoyl which may be substituted in any position with one or more of the groups alkoxy, alkyl, fluorine, chlorine, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2, 3 or 4 and which may be substituted in position
5 with one of the groups fluorine and nitro;
2-thiazolyl which may be substituted in the 4 and/or 5 positions with one or two of the groups alkyl and phenyl;
R3 and R4, being similar or different, are chosen from the group comprising hydrogen; hydroxy; fluorine; alkyl; functionalised alkyl; hydroxyalkyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenylamino and diphenylamino wherein the phenyl groups may be similar or different; aminoalkyl, (alkylamino) alkyl, (dialkylamino) alkyl,
(phenylamino) alkyl and (diphenylamino) alkyl wherein the alkyl groups may be similar or different, and the phenyl groups may be similar or different;
(4-methylmorpholinyl) alkyl; benzoyl which may be substituted in any position with one ore more of the groups alkoxy, alkyl, fluorine, chlorine, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2,3 or 4 and which may be substituted in position
5 with fluorine, chlorine, hydroxy or nitro; alkanoyl; oxoalkyl which may be substituted in any position with alkyl or phenyl; phenylazo; alkenyl; phenyl (phenylimino) methyl; phenyl; 2-furfuryl; lH-indol-3-yl;
( lH-indol-3-yl ) methyl ;
and wherein R3, R4 is the methylidene group =C(R5)R6 wherein R5 and R6 being similar or different, are chosen form the group comprising hydrogen; hydroxy; alkyl which may be completely or partially substituted with fluorine and chlorine; alkenyl which may be substituted with one or more phenyl groups, and which alkenyl may be completely or partially substituted with deuterium, fluorine, chlorine, nitro, phenylthio, phenylsulphinyl and phenylsulphonyl; phenyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenymethyleneamin; 2-furanyl; lH-indol-2-yl; lH-indol-3-yl.
3. Use of a compound according to claim 1 for preparing a composition for treating HIV/AIDS.
4. Use of a compound according to claim 2 for preparing a composition for treating HIV/AIDS.
5. Use of a compound according to claim 1 for preparing a composition for treating measles.
6. Use of a compound according to claim 2 for preparing a composition for treating measles.
7. Use of a compound according to claim 1 wherein the compound is Oxyphenbutazone.
8. Use of Oxyphenbutazone for preparing a composition for treating HIV/AIDS.
9. Use of Oxyphenbutazone for preparing a composition for treating measles.
10. Use of a compound according to claim 7 for preparing a composition wherein Oxyphenbutazone is present in the composition in an amount suitable for delivering an effective dose of Oxyphenbutazone.
11. Use of a compound according to claim 10, wherein
Oxyphenbutazone is present in the range of 0.0075 - 225 μmol/kg body weight.
12. Use of a compound according to claim 10, wherein Oxyphenbutazone is present in the range of 0.075 - 37.5 μmol/kg body weight.
13. Use of a compound according to claim 10, wherein
Oxyphenbutazone is present in the range of 0.15 - 2.25 μmol/kg body weight.
14. Use of a compound according to claim 10 for preparing a composition suitable for being administered in 1 - 3 partial doses per day.
15. Use of a compound according to claim 1 for preparing a composition suitable for being administered systemically, especially enterally, preferably parenterally, such as subcutananeously, intravenously, intradermally and orally, most preferably orally.
16. Use of a compound according to claim 10 for making a composition suitable for being administered systemically, especially enterally, preferably parenterally, such as subcutananeously, intravenously, intradermally and orally, most preferably orally.
17. Use of a compound according to claim 1 for preparing a composition including additionally an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor.
18. Use of a compound according to claim 10 for preparing a composition including additionally an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor.
19. A method of treating a viral infection wherein a compound according to claim 1 is administered to a subject in need of such a treatment.
20. A method according to claim 19 for treating HIV/AIDS.
21. A method according to claim 19 for treating measles.
22. A method according to claim 19 for treating a viral infection wherein a compound according to claim 1 is administered orally to a subject in need of such a treatment.
23.A method according to claim 20 for treating HIV/AIDS wherein a compound according to claim 1 is administered orally to a subject in need of such a treatment.
24.A method according to claim 21 for treating measles wherein a compound according to claim 1 is administered orally to a subject in need of such a treatment.
25.A method of treating a viral infection wherein a compound according to claim 10 is administered to a subject in need of such a treatment.
26.A method according to claim 25 for treating HIV/AIDS
27.A method according to claim 25 for treating measles.
28.A method according to claim 25 for treating a viral infection wherein a compound according to claim 10 is administered orally to a subject in need of such a treatment .
29.A method according to claim 26 for treating HIV/AIDS wherein a compound according to claim 10 is administered orally to a subject in need of such a treatment.
30.A method according to claim 27 for treating measles wherein a compound according to claim 10 is administered orally to a subject in need of such a treatment.
31.A method according to claim 19 wherein a compound according to claim 1 in addition to an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor is aministered.
32. A method according to claim 25 wherein a compound according to claim 10 in addition to an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor is aministered.
AMENDED CLAIMS
[received by the International Bureau on 1 1 August 2000 (1 1.08.00) ; original claims 1-32 replaced by amended claims 1-32 (10 pages)]
Wha t is claimed is :
1. Use of a compound having formula (I) :
Figure imgf000038_0001
or therapeutic acceptable salts thereof,
wherein
R1 and R2, which are similar or different, are chosen from the group comprising hydrogen; alkyl; functionalised alkyl; phenyl; benzoyl which may be substituted in any position with one or more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2, 3 or 4 and which may be substituted in any position, with the exception of position 1, with one or more of the groups alkoxy, alkyl, halogen, hydroxy and nitro;
2-thiazolyl which may be substituted in the 4 and 5 positions with one or two of the groups alkyl and phenyl;
1, 2-penzιsothιazol-2 (3H) -yl; 1, 2-benzisothiazol-2 (3H) -yl S, S-dioxide;
3-oxo-l, 2-benzisothiazol-2 (3H) -yl S,S dioxide;
1H-1, 2, 4-triazol-3-yl which may be substituted in position
5 with one of the groups alkyl and phenyl;
R3 and R4, being similar or different, are chosen from the group comprising hydrogen; hydroxy; halogen; alkyl; functionalised alkyl; hydroxyalkyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenylamino and diphenylamino wherein the phenyl groups may be similar or different; aminoalkyl, (alkylamino) alkyl, (dialkylamino) alkyl,
(phenylamino) alkyl and (diphenylamino) alkyl wherein the alkyl groups may be similar or different, and wherein the phenyl groups may be similar or different;
( 4-methylmorpholinyl) alkyl; benzoyl which may be substituted in any position with one ore more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2,3 or 4 and which may be substituted in any position, except for position 1, by one or more of the groups alkoxy, alkyl, halogen, hydroxy and nitro; alkanoyl; oxoalkyl which may be substituted in any position with alkyl or phenyl; phenylazo; alkenyl which may be completely or partially substituted with deuterium, halogen, nitro, phenylthio, phenylsulphinyl and phenylsulphonyl;
2, 3-dihydro-l, 3-dioxo-lH-indene-2-yl which may be substituted in one or more positions with one or more of the groups alkyl, acyloxy, hydroxy and halogen;
1, 3-dihydro-3-oxo-l-isobenzofuranyl; phenyl (phenylimino) methyl which may be completely or partially deutetrated in any position, or substituted in one or more positions with one or more of the groups alkoxy, alkanoyl, alkyl, halogen, hydroxy and nitro; phenyl;
2-furfuryl; lH-indol-3-yl; (lH-indol-3-yl) methyl;
and wherein R3, R4 is the methylidene group =C(R5)R6 wherein R5 and R6 are similar or different and are chosen form the group comprising hydrogen; hydroxy; alkyl which may be completely or partially substituted with halogen, preferably fluorine and chlorine; alkenyl; phenyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenymethyleneamin;
2-furanyl; lH-indol-2-yl; lH-indol-3-yl;
or are
2, 5-bis [ (3-ethyl-2 (3H) -benzothiazolylidene) ethylidene] - cyclopentyliden; cycloalkylidene with 4 or more C-atoms in the ring; diazo;
4- [ (4-chlorophenyl)methylene] -5-thioxo-3-isothiazol- idinylidene; 1, 5-dihydro-5-oxo-2H-pyrrol-2-ylidene which may be substituted in position 1 with one of the groups phenyl and/or in position 5 with the groups phenylhydrazono or phenylimino; dihydro-5-oxo-2 (3H) -furanylidene : 3, 5-dioxo-4-pyrazolidinylidene;
4 (1H) -quinolinylidene;
2- (3-ethyl-2 (3H) -benzothiazolylidene-1- [ (3-ethyl-2 (3H) - benzothiazolylidene) methyl] ethylidene;
2- (3-ethyl-2 (3H) -benzothiazolylidene-1- [ ( l-ethyl-3, 4- dihydro-2 (1H) -quinolinylidene) methyl] ethylidene;
2- (3-ethyl-2 (3H) -benzothiazolylidene-1- [ (l-ethyl-2 (1H) - quinolinylidene ) methyl ] ethylidene ;
(3-ethyl-2-oxazolidinylidene) ethylidene,
provided that the compound is not butyl-4-diphenyl-l, 2- pyrazolidine-dione-3, 5,
for preparing a pharmaceutical composition for treating viral diseases/infections.
2. Use of a compound according to claim 1 wherein
R1 and R2, which are similar of different, are chosen from the group comprising hydrogen; alkyl; functionalised alkyl; phenyl; benzoyl which may be substituted in any position with one or more of the groups alkoxy, alkyl, fluorine, chlorine, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2, 3 or 4 and which may be substituted in position
5 with one of the groups fluorine and nitro;
2-thiazolyl which may be substituted in the 4 and/or 5 positions with one or two of the groups alkyl and phenyl;
R3 and R4, being similar or different, are chosen from the group comprising hydrogen; hydroxy; fluorine; alkyl; functionalised alkyl; hydroxyalkyl; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenylamino and diphenylamino wherein the phenyl groups may be similar or different; aminoalkyl, (alkylamino) alkyl, (dialkylamino) alkyl,
(phenylamino) alkyl and (diphenylamino) alkyl wherein the alkyl groups may be similar or different, and the phenyl groups may be similar or different; (4-methylmorpholinyl) alkyl; benzoyl which may be substituted in any position with one ore more of the groups alkoxy, alkyl, fluorine, chlorine, hydroxy and nitro; pyridinyl which may be attached to the molecular core in position 2,3 or 4 and which may be substituted in position
5 with fluorine, chlorine, hydroxy or nitro; alkanoyl; oxoalkyl which may be substituted in any position with alkyl or phenyl; phenylazo; al kenyl ; phenyl (phenylimino) methyl; phenyl;
2-furfuryl; lH-indol-3-yl;
( lH-indol-3-yl ) methyl ;
and wherein R3, R4 is the methylidene group =C(R5)R6 wherein R5 and R6 being similar or different, are chosen form the group comprising hydrogen; hydroxy; alkyl which may be completely or partially substituted with fluorine and chlorine; alkenyl which may be substituted with one or more phenyl groups, and which alkenyl may be completely or partially substituted with deuterium, fluorine, chlorine, nitro, phenylthio, phenylsulphinyl and phenylsulphonyl; phenyl ; amino, alkylamino and dialkylamino wherein the alkyl groups may be similar or different, or are 2- (diethylamino) ethyl; phenymethyleneamin;
2-furanyl; lH-indol-2-yl; lH-indol-3-yl.
3. Use of a compound according to claim 1 for preparing a composition for treating HIV/AIDS.
4. Use of a compound according to claim 2 for preparing a composition for treating HIV/AIDS.
5. Use of a compound according to claim 1 for preparing a composition for treating measles.
6. Use of a compound according to claim 2 for preparing a composition for treating measles.
7. Use of a compound according to claim 1 wherein the compound is Oxyphenbutazone.
8. Use of Oxyphenbutazone for preparing a composition for treating HIV/AIDS.
9. Use of Oxyphenbutazone for preparing a composition for treating measles.
10. Use of a compound according to claim 7 for preparing a composition wherein Oxyphenbutazone is present in the composition in an amount suitable for delivering an effective dose of Oxyphenbutazone.
11. Use of a compound according to claim 10, wherein
Oxyphenbutazone is present in the range of 0.0075 - 225 μmol/kg body weight.
12. Use of a compound according to claim 10, wherein
Oxyphenbutazone is present in the range of 0.075 - 37.5 μmol/kg body weight.
13. Use of a compound according to claim 10, wherein
Oxyphenbutazone is present in the range of 0.15 - 2.25 μmol/kg body weight.
14. Use of a compound according to claim 10 for preparing a composition suitable for being administered in 1 - 3 partial doses per day.
15. Use of a compound according to claim 1 for preparing a composition suitable for being administered systemically, especially enterally, preferably parenterally, such as subcutananeously, intravenously, intradermally and orally, most preferably orally.
16. Use of a compound according to claim 10 for making a composition suitable for being administered systemically, especially enterally, preferably parenterally, such as subcutananeously, intravenously, intradermally and orally, most preferably orally.
17. Use of a compound according to claim 1 for preparing a composition including additionally an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor.
18. Use of a compound according to claim 10 for preparing a composition including additionally an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor.
19. A method of treating a viral infection wherein a compound according to claim 1 is administered to a subject in need of such a treatment.
20. A method according to claim 19 for treating HIV/AIDS,
21. A method according to claim 19 for treating measles.
22. A method according to claim 19 for treating a viral infection wherein a compound according to claim 1 is administered orally to a subject in need of such a treatment .
23. A method according to claim 20 for treating HIV/AIDS wherein a compound according to claim 1 is administered orally to a subject in need of such a treatment.
24. method according to claim 21 for treating measles wherein a compound according to claim 1 is administered orally to a subject in need of such a treatment.
25. A method of treating a viral infection wherein a compound according to claim 10 is administered to a subject in need of such a treatment.
26. A method according to claim 25 for treating HIV/AIDS.
27. A method according to claim 25 for treating measles.
28. A method according to claim 25 for treating a viral infection wherein a compound according to claim 10 is administered orally to a subject in need of such a treatment.
29. A method according to claim 26 for treating HIV/AIDS wherein a compound according to claim 10 is administered orally to a subject in need of such a treatment.
30. method according to claim 27 for treating measles wherein a compound according to claim 10 is administered orally to a subject in need of such a treatment.
31. A method according to claim 19 wherein a compound according to claim 1 in addition to an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor is aministered.
2.A method according to claim 25 wherein a compound according to claim 10 in addition to an inhibitor for inhibiting a reverse transcriptase enzyme and/or protease inhibitor and/or an integrase inhibitor is aministered.
PCT/NO2000/000086 1999-03-12 2000-03-10 The use of 3,5-pyrazolidinedione derivatives to combat viral infections WO2000054771A1 (en)

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NO991244A NO991244D0 (en) 1999-03-12 1999-03-12 Use of antiflogistics in inhibiting viral infections

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0237796A2 (en) * 1986-02-14 1987-09-23 Research Development Corporation of Japan New use of prostaglandin biosynthesis inhibitors
EP0285730A1 (en) * 1986-02-04 1988-10-12 Jean-Olivier Miesch Use of 4-butyl-1,2-diphenyl-pyrazolidine-3,5-dione as an antiviral medicament for treating AIDS

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0285730A1 (en) * 1986-02-04 1988-10-12 Jean-Olivier Miesch Use of 4-butyl-1,2-diphenyl-pyrazolidine-3,5-dione as an antiviral medicament for treating AIDS
US4956377A (en) * 1986-02-04 1990-09-11 Miesch Jean Olivier Use of butyl-4-diphenyl-1,2-pyrazolidine-dione-3,5 as an antiviral agent in humans and animals
EP0237796A2 (en) * 1986-02-14 1987-09-23 Research Development Corporation of Japan New use of prostaglandin biosynthesis inhibitors

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