EP1668023A1 - B-l-nucleosides and use thereof as pharmaceutical agents for treating viral diseases - Google Patents

Abstract

The invention relates to nucleoside analogues, nucleic acids and pharmaceutical agents comprising these, and to the use of the nucleoside analogues, nucleic acids and pharmaceutical agents for diagnosing, preventing or treating a viral, bacterial, fungal and/or parasitic infection or cancer, particularly hepatitis infections. The invention also relates to a method for producing the nucleoside analogues, to a kit, and to the use thereof for preventing and treating viral diseases, particularly hepatitis infections.

Description

 β-L nucleosides and their use as pharmaceutical agents for the treatment of viral diseases

description

The invention relates to new β-L-5-methylcytosine nucleosides of the structure as can be seen from the following formulas I and II,

Formula I.

in which

Ri = H, OH, FR ₂ = H, OH R ₃ = HR ₄ = H, OH, N ₃ and R ₅ = H, acetyl, palmitoyl, alkoxycarbonyl, carbamate,

Mean phosphonate, monophosphate, diphosphate or triphosphate, namely

if R _lf R ₂ and R ₃ = H, then R = H, OH or N ₃ ,

-if Ri = OH or F, then R ₂ and R ₃ = H and R ₄ =

OH,

Formula II

in which

R ₆ = H, F and R ₇ = H mean

and their use as pharmaceutical active substances or agents for the prophylaxis and / or treatment of infections which are caused in particular by the hepatitis B virus (HBV). The β-L-5-methylcytosine nucleosides, their acceptable salts, or their prodrugs can be used alone or in combination with other β-L-nucleosides, with 3-deazauridine or with other anti-HBV-active compounds. Areas of application of the invention are medicine and the pharmaceutical industry.

PRIOR ART HBV is the triggering agent for hepatitis B, an infectious disease, the chronic course of which affects about 350 million people worldwide, especially in Southeast Asia, Africa and South America. In many cases, hepatitis B infections lead to fatal outcome from liver function failure. In addition, the chronic course is associated with a greatly increased risk of a primary liver carcino, which in China alone leads to around one million new cases each year. While the exact mechanism by which HBV can induce liver tumors is unknown, it can be assumed that tumor induction is closely related to HBV-induced chronic inflammation, developing cirrhosis and regeneration processes in liver tissue. The genetically engineered vaccine, which has been available for many years, is not suitable for the treatment of hepatitis B because it does not help people who are already infected and cannot stop the chronic course mentioned. In recent years, genetically engineered α-interferon has proven particularly useful in the treatment of HBV infections. It is a cytokine with broad antiviral and immunomodulating activity. However, it is only effective in about 33% of patients, has significant side effects and cannot be given orally. A successfully used nucleoside derivative approved by the US Food and Drug Administration and also in Germany is lamivudine (ß-L-2 ", 3" -dideoxy-3 "~ thiacytidine), also known as thiacytidine (3TC), which is from Liotta et al., In US Patent 5 539 116. It is highly effective in both HbeAg positive and HbeAgegative patients and has few side effects, although a rapid drop in HBV DNA and If the normalization of the alanine transferase activity is found in the serum, the HBV apparently cannot be completely eliminated from the liver under this therapy, so that even after the end of one year of treatment it is possible in many cases that hepatitis B This course is being attempted to be prevented by extending treatment to several years in the hope of being able to completely eliminate HBV (Alberti et al., J Med Virol 2002, 67: 458-462). However, such therapies are increasing

Resistance to lamivudine, which can be 45-55% after the second year of treatment (Liaw et al., Gastroenterology 2000, 119: 172-180).

The development of additional effective compounds is therefore urgently needed to replace monotherapy with combination therapy, which can not only be more effective, but can also significantly reduce the risk of resistance, as has been shown in the long-term treatment of HIV infections (Richman , Nature 410: 995-1000). Lamivudine belongs to a group of so-called ß-L nucleosides. They are mirror images of the naturally occurring ß-D nucleosides and were long considered to be non-enzymatic and therefore ineffective in biological systems.

This dogma was first developed in 1992 by the findings of Spadari et al. relativized, who had discovered that ß-L-thymidine is not converted by the cellular thymidine kinase, but is a substrate of the corresponding enzyme of the herpes simplex virus 1 (Spadari et al., J Med Chem 1992, 35: 4214-4220) , It was later shown that β-L-nucleosides can be substrates or inhibitors not only for some vira'le but also for some cellular enzymes (Review: Maury, Antiviral Chem Chemother 2000, 11: 165-190).

Subsequently, a large number of β-L-nucleoside analogues were synthesized in pure form, among which, in addition to the lamivudine mentioned (3TC; β-L-2 ", 3" - dideoxy-3 "-thia-cytidine, - Jeong et al., J Med Chem 1993, 36: 181-195), emtricitabine (L-FTC; β-L-2 ", 3" -di ~ deoxy ~ 5-fluoro-3 "-thiacytidine; Furman et al., Antimicrob Agents & Chemother 1992, 36: 2686-2692;), β ~ L-2 "~ fluoro-5-methylarabinofuranosyluracil (L-FMAU; Clevudin; Chu et al., Antimicrob Agents & Chemother 1995, 39: 979-981), β-L-2 ", 3" -dideoxycytidine and β-L-2 ", 3" -dideoxy-5-fluorocytidine (L-ddC, L-FddC; Lin et al., J Med Chem 1994, 37: 798- 803), ß-L-2 ", 3" -dideoxy-2 ", 3" -didehydrocytidine (L-ddeC; Lin et al., J Med Chem 1996, 39: 1757-1759), and ß-L thymidine ( L-TdR; Telbivudin; by Janta-Lipinski et al., J. Med. Chem. 1998, 41: 2040-2046; Bryant et al., Antimicrob Agents & Chemother 2001, 45: 229-235) as the most effective and The most promising inhibitors of HBV replication in vitro and in vivo have emerged. T. extremely low cytotoxicity. Among the D-nucleosides is entecavir (BMS 200475), a carbocyclic deoxyguanosine derivative (Innaimo et al., Antimicrob Agents & Chemother 1997, 41: 1444-1448), which is superior to lamivudine in the treatment of hepatitis B in a first clinical study (Lai et al., Gastroenterology 2002, 123: 1831-1838).

Further syntheses of L-nucleosides are described in Mugnaini et al. , Bioorg Med Chem 2003, 11: 357-366; Marquez et al., J Med Chem 1990, 33: 978; Lee et al. , Nucleosides & Nucleotides 1999, 18: 537-540; Faraj et al. , Nucleosides & Nucleotides 1997, 16: 1287-1290; Song et al. , J Med Chem 2001, 44: 3985-3993; Kotra et al. , J Med Chem 1997, 40: 19-44; Choi et al. , Organic Lett 2002, 4: 305-307; Gumina et al., Curr Top Med Chem 2002, 2: 1065-1086; Holy, Tetrahedron Lett. 1971, 189-193; Holy, Collect Czech Chem Commun 1972, 37: 4072-4082, the following patents also describe β-L-nucleosides as potential antivirals: Gosselin et al., US Patent 6,395,716, Schinazi et al, US Patent 20020107221 AI; Chu et al., U.S. Patents: 5,565,438, 5,567,688, 5587362, WO Patent: 92/18517 by Yale University and University of Georgia Research Foundation, Inc.

In addition to the β-L-cytosine nucleosides with unmodified cytosine, as in β-L-deoxycytidine (Bryant et al., Antimicrob Agents & Chemoth 2001, 45: 229-235), β-L-2 ", 3" - dideoxycytidine , (Lin et al., J Med Chem 1994, 37: 798-803), β-L-2 ", 3" -dideoxy-2 ", 3" -didehydrocytidine (Lin et al., J Med Chem 1996, 39: 1757-1759), β-L-2 "-fluoro-arabinofurano-sylcytosine (FAC; Ma et al., J Med Chem 1996, 39: 2835-2843), β-L -Arabinofuranosylcytosine (L-AraC; Chu et al., US Patent 5567688), β-L-2 ", 3" -dideoxy-2 ", 3" - didehydro-2 "-fluorcytidine (L-2"FddeC; Lee et al., J Med Chem 1999, 42: 1320-1328), some 5-modified cytosine derivatives have also been synthesized and investigated, in particular 5-fluorocytosine derivatives, which are either more potent than the compounds with unmodified bases, such as β-L-2 " , 3 "-dideoxy-2", 3 "-dehydro-5-fluorocytidine (L-FddeC; Lin et al., J Med Chem 1996, 39: 1757-1759), as effective as β-L-2", 3 "-Dideoxy-5-fluorocytidine (L-FddC; Lin et al., J Med Chem 1994, 37: 798-803) or β-L-2", 3 "- dideoxy-2", 3 "-dehydro-2 "-fluoro-5-fluorocytidine (L ~ 2"F-dedeFC; Lee et al., J Med Chem 1999, 42: 1320-1328), are less effective than β-L-deoxy-5-fluorocytidine (FdC; Bryant et al., Antimicrob Agents & Chemoth 2001, 45: 229-235) or show no effect on HBV replication, such as ß-L-2 "-fluoro-arabinofuranosyl-5-fluorocytosine (L-FAFC; Ma et al. , J Med Chem 1996, 39: 2835-2843) or β-L-arabinofuranosyl-5-fluorocytosine (L-AraFC; Griffon et al., Eur J Med Chem 2001, 36: 447-460).

The following 5-chloro, bromine and methyl-modified L-cytosine nucleosides have also been described as ineffective or not very effective: β-L-deoxy-5-chlorocytidine (CldC; Bryant et al., Antimicrob Agents & Chemother 2001, 45 : 229-235), ß-L-2 "-fluoroarabinosyl-5-chlorocytosine, ß-L-2" -fluoro-arabinosyl-5-bromocytosine (L-FAC1C, L-FABrC; Ma et al., J Med Chem 1996, 39: 2835-2843), β-L-2 ", 3" -dideoxy-3 "-thia-5-methylcytidine, β-L-2", 3 "-dides-oxy-3" -thia -5-bromocytidine, ß-L-2 ", 3" -dideoxy-3 "-thia-5-chlorocytidine and ß-L-2", 3 "- Dideoxy-3 "-fluoro-5-methylcytidine (Dong et al., Proc Natl Acad Sei USA 1991, 88: 8495-8499; Matthes et al., Unpublished), so that 5-fluorine modifications of β-L -Cytosine nucleosides are known, but other 5 modifications have only been produced in isolated cases and their testing has not had any significant effects on HBV replication.

Some of the L-nucleosides mentioned are not only effective inhibitors of HBV replication, but also of HIV replication. So z. B. Lamivudine also approved for the treatment of HIV infection. Further, already mentioned ß-L-cytosine nucleosides, such as L-ddC, L-5FddC, L-FddeC, FTC are also strong inhibitors of HIV replication, the importance of which for therapy lies in new effective compounds for combination therapy To have available and to be able to counter possible development of resistance (Menendez-Arias, Trends Pharmacol Sei 2002, 23: 381-388). There are also a number of ß-L nucleosides that only replicate HBV (e.g. L-FMAU, L-TdR, L-CdR, L-3 "FddC, L-ddeC), others that only Inhibit HIV replication (e.g. abacavir).

All of the β-L-nucleosides mentioned are taken up by the HBV- or HIV-infected cells and have to be converted by the cell's own enzymes to the nucleoside triphosphates. Only in this form can they bind their actual target, the HBV-DNA polymerase, or the reverse transcriptase in competition to the normal substrates and exert a strong inhibition. As a result, the viral genomes can no longer be synthesized and virus production comes to a standstill. This inhibition must be selective, ie, it must remain restricted to the viral polymerases and must not include the cellular DNA polymerases, because otherwise the proliferation of rapidly proliferating cells would be impaired as a result of the inhibition of the synthesis of cellular DNA.

The invention is based on the object of developing new antivirally active β-L-methylcytosine nucleosides which are particularly effective against hepatitis B virus infections and HIV infections and which, with good tolerability and low toxicity, are highly effective against this infection.

Surprisingly show ß-L-5-methylcytosine nucleosides according to formula I or formula II

Formula I.

in which

Ri = H, OH, F

R ₂ = H, OH

R ₃ = H

R ₄ = H, OH, N ₃ and R ₅ = H, acetyl, palmitoyl, alkoxycarbonyl, carbamate,

Phosphonate, monophosphate, Mean diphosphate or triphosphate, namely,

-if Ri, R ₂ and R ₃ = H, then R ₄ = H, OH or N ₃ ,

-when R _x = OH or F, then R ₂ and R ₃ = H and R ₄

OH,

Formula II

in which

R ₆ = H, F and R ₇ mean H,

high antiviral activity, especially against HBV. Particularly effective are: ß-L-5-methyldeoxycytidine (ß-L-MetCdR), ß-L-2 ", 3" -dideoxy-5-methylcytidine (ß-L-ddMetC), ß-L-2 ", 3 "-Didehydro-2", 3 "-dideoxy-5-methylcytidine (ß-L-ddeMetC), ß-L-arabinofuranosyl-5-methylcytosine (ß-L-AraMetC), ß-L-2", 3 "- Didehydro-2 ", 3" -dideoxy-2 "-fluoro-5-methylcytidine (ß-L-FddeMetC), ß-L- 2 "-fluoroarabinofuranosyl-5-methylcytosine (ß-L-FMAC) and ß-L-3" -azido-2 ", 3" -dideoxy-5-methylcytidine (LN ₃ MetCdR), the effectiveness of which by simultaneous application of 3-deazauridine can be greatly increased.

In contrast to the ß-L series, some 5-methylcytidine derivatives of the compounds mentioned are known in the ß-D series. The triphosphates of ß-D-AraMetC, ß-D-ddeMetC, ß-D-FMetC, ß-D-AzMetC have proven to be highly effective inhibitors of HBV DNA polymerase, (Matthes et al., Antimicrob Agent & Chemother 1991, 35: "1254-1257), but the nucleosides of which are intracellular and only moderately effective in vivo (Matthes et al., Biochemical Pharmacol 1992, 43: 1571-1577), which is probably a result of the high rate of deamination by the deoxycytidine deaminase, which leads to the fact that intracellularly almost exclusively thymidine analogues exist for the enantiomeric β-L-deoxycytidine and a large number of its modified derivatives (eg β-L-AraC, 3TC, β-L-FTC, β-L-FddC, ß-L-FddeC), on the other hand, it could be shown that such deamination no longer occurs at the nucleoside level (Review: Maury, Antiviral Chem Chemother 2000, 11: 165-190) and thus the first and most difficult activation step of ß-L-5 -Methylcytosine nucleosides to monophosphate a task of stereospecific hen deoxycytidine kinase and not thymidine kinase 1, which can hardly phosphorylate L-thymidine derivatives (Focher et al. , Current Drug Targets-Infectious Disorders 2003, 3: 41-54). 3-deazauridine activates cellular deoxycytidine kinase and its intracellularly formed triphosphate is also able to inhibit cellular CTP synthase (Gao et al., Nucleosides Nucleotides Nucleic Acids 2000, 19: 371-377). These two effects on the cellular deoxycytidine metabolism have the consequence that 3-deazauridine leads to higher triphosphate levels of the β-L-5-methyl-cytosine nucleosides according to the invention and their effectiveness against HBV replication is thereby greatly increased.

It has surprisingly been found that the nucleosides according to the invention, ie the β-L-methylcytosine nucleosides, can be used with a high antiviral activity against selected viruses, in particular against hepatitis viruses, preferably against hepatitis B viruses. The nucleosides or nucleoside analogs according to the invention are structures which differ in some features from the naturally occurring nucleosides, but there is an analogy to naturally occurring nucleosides in at least two essential points. On the one hand, a - possibly also modified - nucleobase is always required, which is necessary as a binding site to the complementary viral DNA parent strand. On the other hand, there must be a functional group in the former 5 "position which allows the formation of an energy-rich triphosphate from the nucleosides according to the invention or the nucleoside analogues. When the viral enzyme DNA polymerase is inhibited, the nucleoside triphosphate can only be inhibited in the However, direct administration of the active nucleoside triphosphate as an active ingredient is less preferred, since these are broken down in the blood plasma by non-specific phosphatases to the corresponding free nucleoside analogues. In addition, due to their negative charge, triphosphates cannot cross a cell membrane and thus cannot reach the site of action within the cell. Some viruses, such as herpes viruses, are able, with their own viral thymidine kinase, to metabolize the nucleoside analogues according to the invention in infected cells to the respective nucleoside monophosphate, which in turn changes from the cell's own enzyme to triphosphate, the actual active metabolite. is converted.

The triphosphate competes as an alternative substrate with the natural substrate for incorporation into the DNA. In particular, the lack of a 3 "hydroxyl function or an analog group prevents further chain extension. However, it is also possible that the triphosphate acts as a competitive inhibitor of the viral DNA polymerase. It is so in many ways possible that the replication of the

Virus is disrupted or completely prevented.

In a preferred embodiment of the invention, derivatives of the nucleosides according to the invention are used. These can be structures which have modifications which in particular increase the antiviral activity. However, it can also be a salt, a phosphonate, a monophosphate, a diphosphate, a triphosphate, an ester or a salt of such esters. Advantageously, such compounds can be used effectively in antiviral prophylaxis and therapy and have little or no side effects. In a preferred embodiment of the invention, the 5-methylcytosine-β-L-nucleosides according to the invention are β-L-5-methyldeoxycytidine, 5-methylcytosine, β-L- 2 ", 3" -dideoxy-5-methylcytidine ( L-ddMetC), ß-L-2 ", 3" - didehydro-2 ", 3" -dideoxy-5-methylcytidine (L-ddeMetC), ß-L-arabinofuranosyl-5-methylcytosine (L-MetaraC), ß -L-2 "-Fluoro-arabinofuranosyl-5-methylcytosine (L-FMAC), ß-L-2", 3 "-Dide- hydro-2", 3 "-dideoxy-2" -fluoro-5-methylcytidine ( L-FddeMetC) and / or ß-L-3 "-azido-2", 3 "-dideoxy-5-methylcytidine (L-AzMetdC), the C-5 OH group of the sugar components being replaced by O-acetyl, O- Palmitoyl, alkoxycarbonyl, phosphonate, mono-, di-, triphosphate or another protective group can be replaced and converted into the hydroxyl group in a subsequent reaction, with which these modifications represent so-called prodrugs of the β-L-5-methylcytosine nucleosides, which advantageously have a particularly high antiviral activity.

Β-L-5-methyl-deoxycytidine (L-MetCdR), β-L-2 ", 3" -dideoxy-5-methylcytidine (L-ddMetC), β-L-2 ", 3" -didehydro-2 are particularly preferred ", 3" -dideoxy-5-methylcytidine (L-ddeMetC), ß-L-arabinofuranosyl-5-methylcytosine (L-AraMetC) and ß-L-2 ", 3" -idehydro-2 ", 3" -dideoxy -2 "- fluoro-5-methylcytidine (L-FddeMetC), but also ß-L-2" - fluoroarabinofuranosyl-5-methylcytosine (L-FMAC) and ß-L-3 "- azido-2", 3 "- dideoxy-5-methylcytidine (L-AzMetdC).

The compounds according to the invention are prepared by processes known per se by condensation of the sugar part and heterocycle or by modification of the L-ribosyl radical.

The preferred compounds are particularly suitable for

Treatment of the clinical manifestation of viral hepatitis B infection in humans as well as prophy lactic treatment for hepatitis infections. The preferred compounds are particularly effective in inhibiting the multiplication of DNA viruses at the level of virus-specific transcription or translation. In particular, the substances can influence the multiplication of viruses by inhibiting the enzyme reverse transcriptase or by breaking the growing DNA chain. Furthermore, the structures according to the invention can cause the separation of a base pair and thus a wrong association or structural shift in the growing DNA chain or they prevent the formation of an RNA-DNA hybrid and can thus lead to chain termination or to the inhibition or modification of the viral replica - ions. If selected structures of the invention are not chain terminators, the incorporation of a nucleoside according to the invention into the DNA by the viral transcriptase can have an inhibiting effect because the nucleoside according to the invention introduces multiple mutations into the subsequent polymerization and accumulation cycles, various such mutations for inhibiting the virus to lead. However, it is also preferred that the structures according to the invention - insofar as they are not a chain terminator - or incorporated into the DNA, produce an inhibitory effect by binding to the active or allosteric binding site of the reverse transcriptase and thereby a competing, a non- competitive or non-competitive inhibition. The nucleosides according to the invention naturally had a very broad therapeutic spectrum. It is possible, for example, that they act synergistically in connection with other therapeutic, preferably antiviral, agents by increasing the therapeutic effect additively or non-additively, in particular by increasing the therapeutic index and / or that of each individual compound outbound Reduce risk of toxicity. Accordingly, the nucleosides according to the invention can preferably also be used in combination therapies, including a wide variety of combinations with known therapeutic agents and pharmaceutically acceptable carriers. Of course, veterinary applications are also possible, as are feed additives for all vertebrates. Use in humans is particularly preferred. According to these statements, the nucleosides according to the invention can be used particularly preferably for use as medicaments. The nucleosides can be used alone, as a salt or derivative or as a composition. Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrogen chloride, hydrogen bromide, sulfur, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, milk, salicylic, amber, p-toluenesulfonic, wine, vinegar, Citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids. Preferred acids include hydrochloric, sulfuric, methanesulfonic and ethanesulfonic acids. Methanesulfonic acid is most preferred. Other acids, such as oxalic acid, although not themselves pharmaceutically acceptable, can be used in the preparation of salts which are useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.

Salts derived from appropriate bases include alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium), ammonium and N- (C ^ alkyl) ₄ ⁺ - salts. Combinations of substituents and variables presented by this invention are preferably those that result in the formation of stable compounds. The term "stable" as used herein refers to compounds which have a stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time for the purposes described in detail herein to be useful (for example, therapeutic or prophylactic administration to a mammal or for use in affinity chromatography applications). Typically, such compounds are stable at a temperature of 40 ° C or less, in the absence of moisture or other chemically reactive conditions, for at least a week.

The compounds of the present invention can be used in the form of salts derived from inorganic or organic acids. Included among such acid salts are, for example, the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glycophosphate, gluco-sulfate Heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palate, pectinate, persulfate, 3-phenylpropionate, picrate, piquate, thiocyanate, propionate , Tosylate and undecanoate.

The invention also relates to nucleic acids which contain one or more nucleosides according to the invention as a building block. Such nucleic acids can be prepared by methods known to the person skilled in the art, it is preferred that the nucleic acids according to the invention are composed of 2 to 5000, preferably 10 to 100, nucleoside units. particularly preferably from 20 to 40 nucleoside building blocks. Like the nucleosides according to the invention, the nucleic acids according to the invention can preferably be used as antiviral, antibacterial or fungicidal agents, preferably as antiviral agents, in particular against hepatitis infections. As anti-sense nucleic acids, the nucleic acids according to the invention hybridize with the DNA of the virus and thus inhibit the transcription of the virus DNA. The nucleic acids can be used in particular as agents against hepatitis B, but also herpes, HIV and others, since they can advantageously be broken down only to a limited extent or with difficulty by the cell's own restriction enzymes.

The synthetic nucleic acids or anti-sense nucleic acids of the invention may be in the form of a therapeutic composition or formulation that can be used to inhibit DNA replication in a cell and in the treatment of human hepatitis infections and accompanying diseases in an animal. They can be used as part of a pharmaceutical composition in combination with a physiologically and / or pharmaceutically acceptable carrier. The properties of the vehicle will depend on the route of administration. Such a composition can contain, in addition to the synthetic nucleic acid and the carrier, diluents, fillers, salts, buffers, stabilizers, solvents and other known materials. The pharmaceutical composition according to the invention can also contain other active factors and / or substances which increase the inhibition of HBV expression. For example, combinations of synthetic nucleic acids, each directed against a different region of the HBV nucleic acid, can be used in the pharmaceutical compositions according to the invention. The pharmaceutical composition according to the invention can also other chemotherapeutic agents for the treatment of liver cancer. Such additional factors and / or substances can be incorporated into the pharmaceutical composition in order to produce a synergistic effect with the synthetic nucleic acids according to the invention or to reduce side effects of the synthetic nucleic acids according to the invention. On the other hand, the synthetic nucleic acids according to the invention can be incorporated into formulations of a specific anti-HBV or anti-cancer factor and / or substance for reducing the side effects of the anti-HBV factor and / or substance.

The pharmaceutical composition according to the invention can be in the form of a liposome in which the synthetic nucleic acids according to the invention, in addition to other pharmaceutically acceptable carriers with phiphatic substances such as lipids, which are in an aggregation form as micelles, insoluble monolayers, liquid crystals or lamella layers which are in a aqueous solution are combined. Suitable lipids for a liposomal formulation include, but are not limited to, monoglycerides, diclycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids and the like. Such liposomal formulations are prepared in a manner known per se and are known to the person skilled in the art. The pharmaceutical composition of the invention may further contain other lipid carriers such as lipofectamine or cyclodextrins and the like, thereby enhancing the delivery of the nucleic acids to the cells, or may contain sustained release polymers.

The invention also relates, if appropriate, to a pharmaceutical composition which comprises at least one nucleoside according to the invention and / or a nucleic acid according to the invention together with customary auxiliaries, preferably carriers, adjuvants and / or vehicles. A pharmaceutical agent in the sense of the invention is any agent in the field of medicine that can be used in the prophylaxis, diagnosis, therapy, follow-up or follow-up treatment of patients who have come into contact with viruses, including hepatitis viruses, in such a way that at least temporarily established a pathogenic modification of the overall condition or the condition of individual parts of the organism. For example, it is possible for the pharmaceutical agent in the sense of the invention to be a vaccine, an immunotherapeutic or an immunoprophylactic. The pharmaceutical agent in the sense of the invention can comprise the nucleosides according to the invention or the nucleosic acids according to the invention and / or an acceptable salt or components thereof. These can be, for example, salts of inorganic acids, such as phosphoric acid, or salts of organic acids. It is also possible that the salts are free from carboxyl groups and have been derived from inorganic bases, such as sodium, potassium, ammonium, calcium or iron hydroxides, or from organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol , Histidine and others. Examples of liquid carriers are sterile aqueous solutions which do not comprise any further materials or active ingredients, such as water or those which comprise a buffer such as sodium phosphate with a physiological pH or a physiological saline solution or both, such as phosphate - buffered sodium chloride solution. Other liquid carriers may include more than just a buffer salt, such as sodium and potassium chloride, dextrose, propylene glycol, polyethylene glycol, or others. Liquid compositions of the pharmaceutical compositions can additionally comprise a liquid phase, also with the exclusion of water. Examples of such additional liquid phases are glycerol, vegetable oils, organic esters or water-oil emulsions. The pharmaceutical composition or the pharmaceutical composition typically contains at least 0.1% by weight of the nucleosides or nucleic acids according to the invention, based on the entire pharmaceutical composition. The respective dose or the dose range for the administration of the pharmaceutical agent according to the invention is large enough to achieve the desired prophylactic or therapeutic anti-viral effect. The dose should not be chosen so that undesirable side effects dominate. In general, the dose will vary with the age, constitution, gender of the patient and, of course, the severity of the disease. The individual dose can be set in relation to the primary disease as well as in relation to the occurrence of additional complications. The exact one

Dose can be determined by a person skilled in the art using known means and methods, for example by determining the virus titer depending on the dose or depending on the vaccination schedule or the pharmaceutical carrier and the like. The dose can be selected individually depending on the patient. For example, a dose of the pharmaceutical agent that is still tolerated by the patient can be one whose range in the plasma or in individual organs is locally in the range from 0.1 to 10000 μM, preferably between 1 and 100 μM. Alternatively, the

Dose can also be calculated in relation to the patient's body weight. In such a case, for example, a typical dose of the pharmaceutical agent would have to be set in a range between 0.1 μg to 100 μg per kg body weight, preferably between 1 and 50 μg7kg. However, it is also possible to determine the dose not in relation to the entire patient, but in relation to individual organs. This would be the case, for example, if the pharmaceutical agent according to the invention is placed, for example in a biopolymer, introduced into the respective patient, near certain organs by means of an operation. Several biopolymers are known to the person skilled in the art which can release nucleosides or nucleic acids in a desired manner. Such a gel can contain, for example, 1 to 1000 μg of the compounds according to the invention or the pharmaceutical agent per ml of gel composition, preferably between 5 to 500 μg / ml and particularly preferably between 10 and 100 mg / ml. In such a case, the therapeutic agent is administered as a solid, gel-like or liquid composition.

The pharmaceutical agent can preferably further comprise one or more additional agents from the group of antiviral, fungicidal or antibacterial agents and / or immune stimulators. The antiviral agent is preferably protease inhibitors and / or reverse transcriptase inhibitors. The immune stimulators are preferably bropirimine, anti-human alpha-interferon antibodies, IL-2, GM-CSF, interferons, diethyldithiocarbamate, tumor necrosis factors, naltrexone, tuscarasol and / or rEPO.

In a further preferred embodiment of the invention, the carriers are selected from the group comprising fillers, extenders, binders, humectants, disintegrants, solution retarders, absorption accelerators, wetting agents, adsorbents and / or lubricants. The fillers and extenders are preferably starches, milk sugar, cane sugar, glucose, mannitol and silica, the binder, preferably carbomethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, the humectant, preferably glycerol, and the disintegrant, preferably Agar-agar, calcium carbonate and sodium carbonate, in the solution retarder preferably paraffin and in the absorption accelerator preferably quaternary ammonium compounds, in the wetting agent preferably cetyl alcohol and glycerol monostearate, in the adsorbent preferably kaolin and bentonite and in the lubricant preferably talc, calcium and magnesium stearate and solid polyethylene glycols or mixtures of the listed substances.

The invention also relates to vectors, cells and / or

Organisms which have a nucleoside according to the invention, a nucleic acid according to the invention and / or a pharmaceutical agent according to the invention.

The invention also relates to the use of the nucleosides according to the invention, the nucleic acids according to the invention and / or the pharmaceutical composition according to the invention in the prophylaxis or therapy of a viral, bacterial, fungicidal and / or parasitic infection or cancer. For example, the person skilled in the art is aware that viruses can trigger various tumors. The triggering of these tumors can be prevented prophylactically or treated therapeutically with the compounds according to the invention. It is of course also possible to use the structures according to the invention, for example, in an anti-cancer combination therapy. The person skilled in the art is furthermore aware that, in addition to the viruses, the bacteria associated with the viral diseases or occurring in isolation represent a medical problem. numerous Bacteria are resistant to the known antibacterial agents. The compounds according to the invention can also be used for the prophylaxis and treatment of bacterial infections. Furthermore, the compounds according to the invention can also be used for the production of medicaments for the treatment and prophylaxis of bacterial infections. These can preferably be bacteria of the genera Escherichia coli, Salmonella spp., Shigella flexneri, Citrobacter freundii, Klebsiella pneumoniae, Vibrio spp., Haemophilus influenzae, Yersinia enterolitica, Pasturella haemolytica, and Proteus spp. act.

In a further preferred embodiment of the invention, the invention relates to the use of the compounds according to the invention for preventing the incorporation of other nucleosides during transcription in a growing DNA chain, for preventing the formation of a DNA-RNA hybrid, for separating a base pair or for competing inhibition of a growing DNA chain.

In a further preferred embodiment of the invention, the compounds according to the invention are used to prophylactically or therapeutically treat viral diseases which are combined with one of the following viruses or a combination thereof: hepatitis virus, HIV, bovine immunodeficiency virus, human T- Cell Leukemia Virus, Fine Immunodeficiency Virus, Caprines Arthritis Encephalitis Virus, Equine Infectious Anemia Virus, Ovine Maedi-Visna Virus, Visna-Lentivirus and others. DNA viruses are preferably treated. It is known to the person skilled in the art that such viral infections can occur in combination with bacterial, fungicidal, parasitic or other infections. Use is particularly preferred when the hepatitis virus is a hepatitis B or a hepatitis D virus.

It is furthermore particularly preferred that the pharmaceutical agent according to the invention comprises HBV DNA polymerase inhibitors. Of course, it is also possible that the pharmaceutical agent for the treatment - in particular of Hepatistis B - contains other effective anti-HBV agents, preferably PMEA (adefovir dipivoxil), famciclovir, penciclovir, diaminopurine dioxolane (DAPD), clevudine (L-FMAU ), Entecavir, interferon or thymosin 1 and / or inhibitors of nucleocapsid formation, especially heteroaryl pyrimidines.

It is further preferred that the agents are pegylated.

In addition, it is particularly preferred that the agent contain additional agents which are able to switch off the function of cellular proteins which are essential for the multiplication of HBV.

It is also particularly preferred that it comprises agents against viruses which are resistant to lamivudine or another cytosine nucleoside, such as e.g. B. Emtricitabine (L-FTC), L-ddC or L-ddeC.

This agent can preferably also be used against liver cancer diseases which were triggered by chronic hepatitis, in particular by HBV.

It is further preferred that the β-L-nucleosides increase the effect of other pharmaceutical agents, non-additive, additive or synergistically increased the therapeutic index and / or that of the Reduce the risk of toxicity associated with each compound.

A preferred HIV in the sense of the invention is HIV-1 with subtypes A to J (HIV-1 group M) according to the subtype subdivisions of the prior art and the remotely related HIV-0 (HIV-1 group O). Preferred main subtypes are 1A, IB, IC and ID. The subtypes 1E, IG and 1H are closely related to HIV-IA and are also preferred. The preferred HIV-IA and IC as well as IB and ID show homologies with one another. The likewise preferred HIV-0 is more heterogeneous in the individual virus isolates than HIV-1. A division into subtypes cannot be made. Also preferred is HIV-2, which can be divided into subtypes A to E. It has a milder pathogenicity than HIV-1 and has therefore spread more slowly. The genetic variability leads to changes in the external coat proteins. The influence on cell tropism and the question of how far this is associated with different transmission probabilities has not been sufficiently clarified. Treatment of double infections with different subtypes (e.g. B and E) is also preferred.

In a preferred embodiment of the invention, the nucleosides according to the invention are used in combination with 3-deazauridine. Combined use can mean simultaneous or delayed administration. The combined administration can take place, for example, in a combination agent.

The combination means in the sense of the invention can, for example, be such that in a solution or in a solid such as a tablet, nucleosides according to the invention and 3-deazauridine are contained together. The ratio of nucleosides according to the invention and 3-deazauridine can vary freely. A ratio of nucleosides according to the invention and 3-deazauridine in the range from 1: 10,000 to 10,000: 1 is preferred. Within this range, the ratio of nucleosides according to the invention and 3-deazauridine can vary depending on the desired application. Of course, the at least two components - nucleosides according to the invention and 3-deazauridine - can also be introduced together into a solution or a solid so that they are released with a time delay. However, the combination agent in the sense of the invention can also consist of two separate solutions or two separate solids, the one solution or one solid essentially comprising 3-deazauridine and the other solution or the other solid essentially comprising nucleosides according to the invention. It is possible that the two solutions or solids are associated with a common or with separate carriers. The two solutions and / or the two solids can be present, for example, in a capsule as a common carrier. Such a formulation of the combination agent according to the invention is particularly advantageous when the nucleosides and the 3-deazauridine administration according to the invention are to be carried out with a time lag. This means that the organism is first brought into contact with the nucleosides according to the invention, for example by infusion or by oral administration, in order then to be brought into contact with the other constituent of the combination agent at a later time. Of course, it is also possible for the combination agent to be provided using conventional pharmaceutical methods and processes in such a way that the organism first with 3-deazauridine and then with the nucleosides according to the invention are brought into contact. It is therefore preferred to sequentially bring the organism into contact with the components of the combination agent. The time period between the administration of the two components of the combination agent according to the invention or the first release of nucleosides or 3-deazauridine according to the invention depends on the age, gender, overall constitution of the patient, the disease or other parameters, for example by the treating doctor can be determined by preliminary tests.

In a particularly preferred embodiment of the invention, the compounds according to the invention are used as a prodrug, as a feed additive and / or as a drinking water additive, the use as a feed additive and / or as a drinking water additive being preferred in veterinary medicine.

The use of the compound according to the invention as a prodrug is particularly preferred. The use of endocytosis for the cellular active substance intake of polar compounds is very effective for some, particularly long-lived substances, but it is very difficult to transfer them to a more general application. An alternative to this is the prodrug concept which is well known to the person skilled in the art. By definition, a prodrug contains its active ingredient in the form of an inactive precursor metabolyte. One can differentiate between partially multi-part carrier prodrug systems and biotransformation systems. The latter contain the active ingredient in a form that requires chemical or biological metabolism. Such prodrug systems are known to the person skilled in the art, for example valacyclovir as a precursor of acyclovir or others. Carrier-prodrug systems contain the active substance as such, bound to a masking group that stands out can split off the simplest possible controllable mechanism. The function according to the invention of masking groups in the nucleosides according to the invention is the neutralization of the negative charge on the phosphate residue for improved cell uptake. If the nucleosides according to the invention are used with a masking group, this can also influence other pharmacological parameters, such as oral bioavailability, tissue distribution, pharmacokinetics and stability to non-specific phosphatases. The delayed release of the active ingredient can also have a depot effect. Modified metabolism can also occur, resulting in higher drug efficiency or organic specificity. In the case of a prodrug formulation, the masking group or a linker group which binds the masking group to the active ingredient are selected such that the nucleoside prodrug has sufficient hydrophilicity to be dissolved in the blood serum, sufficient chemical and enzymatic Has stability to get to the site of action and has such hydrophilicity that it is suitable for diffusion-controlled membrane transport. Furthermore, it should enable a chemically or enzymatically induced release of the active substance within a reasonable period of time and of course the released auxiliary components should not have any toxicity. For the purposes of the invention, however, the nucleoside can also be regarded as a prodrug without a mask or a linker and a mask, since the viral DNA polymerase-inhibiting structure is an energy-rich triphosphate, which is first extracted from the incorporated nucleoside by enzymatic and biochemical processes Cell must be provided. In a further preferred embodiment of the invention, the compounds according to the invention are used as a gel, powder, powder, tablet, slow-release tablet, premix, emulsion, infusion formulation, drops, concentrate, granules, syrup, pellet, bolus, capsule, aerosol, spray and / or Inhaled prepared and / or used in this form. The tablets, dragées, capsules, pills and granules can be provided with the usual coatings and casings, optionally containing opacifying agents, and can also be composed such that they release the active ingredient (s) only or preferably in a certain part of the intestinal tract, possibly with a delay , whereby polymer substances and waxes can be used as embedding compositions.

The pharmaceutical compositions of this invention can preferably be used for oral administration in any orally acceptable dosage form, including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers that are used frequently include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweeteners and / or flavors and / or colorants can be added.

The active ingredient (s) can optionally also be in microencapsulated form with one or more of the above-mentioned excipients. In addition to the active ingredient or ingredients, suppositories can contain the usual water-soluble or water-insoluble carriers, for example polyethylene glycols, fats, for example cocoa fat and higher esters (for example C ₁₄ ~ alcohol with C ₁₆ fatty acid) or mixtures of these substances).

In addition to the active ingredient (s), ointments, pastes, creams and gels can contain the usual excipients, for example animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures of these substances ,

In addition to the active ingredient (s), powders and sprays can contain the usual carriers, for example lactose, talc, silica, aluminum hydroxide, calcium silicate and polyamide powder or mixtures of these. Sprays can also contain the usual blowing agents, for example chlorofluorocarbons.

In addition to the active ingredient (s), solutions and emulsions can include the customary carriers such as solvents, solubilizers and emulsifiers, for example water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular Contain cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerin, glycerinfor al, tetrahydofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances. For parenteral administration, the solutions and emulsions can also be in sterile and blood isotonic form.

In addition to the active ingredient (s), suspensions can contain the usual excipients such as liquid diluents Examples include water, ethyl alcohol, propylene glycol, suspending agents, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar agar and tragacanth or mixtures of these substances.

The medicaments can be in the form of a sterile injectable preparation, for example as a sterile injectable aqueous or oily suspension. This suspension can also be formulated using methods known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally compatible diluent or solvent, for example as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are usually used as a solvent or suspending medium. Any mild non-volatile oil, including synthetic mono- or diglycerides, can be used for this purpose. Fatty acids, such as oleic acid and its glyceride derivatives, can be used in the manufacture of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated forms. These oil solutions or suspensions can also contain a long-chain alcohol such as Ph.Helv., Or a similar alcohol as a diluent or dispersant.

The formulation forms mentioned can also contain colorants, preservatives and additives which improve the smell and taste, for example peppermint oil and eucalyptus oil, and sweeteners, for example saccharin. The active Substances of the formula (I) or (II) should preferably be present in the listed pharmaceutical preparations in a concentration of approximately 0.1 to 99.5, preferably approximately 0.5 to 95% by weight of the total mixture.

In addition to the compounds of the formula (I) or (II), the listed pharmaceutical preparations can also contain further active pharmaceutical ingredients. The pharmaceutical preparations listed above are prepared in a customary manner by known methods, for example by mixing the active ingredient (s) with the carrier (s).

The preparations mentioned can be used in humans and animals either orally, rectally, parenterally (intravenously, intramuscularly, subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powder, ointment, drops) and for the treatment of infections in cavities, body cavities. Injection solutions, solutions and suspensions for oral therapy, gels, infusion formulations, emulsions, ointments or drops can be considered as suitable preparations. For local therapy, ophthalmic and dermatological formulations, silver and other salts, ear drops, eye ointments, powder or solutions can be used. In animals, suitable formulations can also be ingested through feed or drinking water. ^" Gels, powders, powders, tablets, prolonged-release tablets, premixes, concentrates, granules, pellets, tablets, boluses, capsules, aerosols, sprays, inhalants can also be used in humans and animals. Furthermore, the compounds according to the invention can be used in other carrier materials such as for example plastics, (plastic chains for local therapy), collagen or bone cement. In a further preferred embodiment of the invention, the compounds according to the invention, ie the nucleoside according to the invention, the nucleic acid according to the invention, the pharmaceutical compositions or vectors according to the invention, cells and organisms in a concentration of 0.1 to 99.5, preferably 0 , 5 to 95, particularly preferably from 20 to 80 wt .-% in a preparation. That is, the compounds according to the invention are present in the pharmaceutical preparations listed above, for example tablets, pills, granules and others, preferably in a concentration of 0.1 to 99.5% by weight of the total mixture. The active ingredient narrow, i.e. the amount of a compound according to the invention, which is combined with the carrier materials to produce a single dosage form, will be able to vary by the person skilled in the art depending on the host to be treated and the particular mode of administration. After the condition of a host or a patient has improved, the proportion of the active compound in the preparation can be changed so that a maintenance dose is available. The dose or frequency of administration, or both as a function of symptoms, can then be reduced to a level at which the improved condition is maintained. If the symptoms have been relieved to the desired level, treatment should stop. However, patients may need long-term interruption treatment after any recurrence of disease symptoms. Accordingly, the proportion of the compounds, that is to say their concentration, in the overall mixture of the pharmaceutical preparation as well as their composition or combination is variable and can be modified and adapted by the person skilled in the art on the basis of his specialist knowledge.

It is known to the person skilled in the art that the compounds according to the invention have an organism, preferably a human or an animal, can be brought into contact in various ways. It is also known to the person skilled in the art that in particular the pharmaceutical compositions can be administered in different dosages. The application should be carried out in such a way that the viral disease is combated as effectively as possible or the outbreak of such a disease is prevented by prophylactic administration. The concentration and the type of application can be determined by a person skilled in the art through routine tests. Preferred applications of the compounds according to the invention are oral application in the form of powder, tablets, juice, drops, capsules or the like, rectal application in the form of suppositories, solutions and the like, parenterally in the form of injections, infusions and solutions, inhalation of Vapors, aerosols and dusts and plasters as well as locally in the form of ointments, plasters, envelopes, rinses and the like. The contacting of the compounds according to the invention is preferably carried out prophylactically or therapeutically. In the case of prophylactic administration, the infection with the viruses mentioned should at least be prevented in such a way that, after individual viruses have penetrated, for example into a wound, further multiplication thereof is greatly reduced or that viruses which have penetrated are almost completely killed off. In the case of therapeutic contact, the patient is already infected and the viruses already in the body are said to either be killed or to be prevented from multiplying. Further preferred forms of application for this are, for example, subcutaneous, sublingual, intravenous, intramuscular, intraperitoneal and / or topical.

The suitability of the selected application forms as well as the

Dose, the application scheme, the adjuvant selection and the like can be obtained, for example, by removing Serum alliquots from the patient, i.e. human or animal, and testing for the presence of viruses, i.e. determining the virus titer, are determined in the course of the treatment protocol. Alternatively and accompanying this, the condition of the liver, but also the amount of T cells or other cells of the immune system, can be determined in a conventional manner to provide an overview of the patient's immunological constitution and in particular the constitution of metabolically important organs. especially the liver. In addition, the clinical condition of the patient can be observed for the desired effect, in particular the anti-infectious, preferably the antiviral effect. Since - as already described - hepatitis in particular, but also HIV or other diseases can be associated with further, for example bacterial or fungicidal, infections or tumor diseases, it is also possible to additionally clinically follow the course of these accompanying infections or tumor diseases. If insufficient antiviral effectiveness is achieved, the patient can be modified and treated further with agents according to the invention or other known medicaments, from which an improvement in the overall constitution can be expected. Of course, it is also possible to modify the carriers or vehicles of the pharmaceutical agent or to vary the route of administration. In addition to oral intake, it can then be provided, for example, that injections, for example intramuscularly or subcutaneously or into the blood vessels, are a further preferred route for the therapeutic administration of the compounds according to the invention. At the same time, delivery via catheters or surgical tubes can also be used. In addition to the concentrations already stated when using the compounds according to the invention, in a preferred embodiment the compounds can also be used in a total amount of 0.05 to 500 mg / kg body weight per 24 hours, preferably from 5 to 100 mg / kg body weight. This is advantageously a therapeutic amount that is used to prevent or improve the symptoms of a disorder or responsive, pathologically physiological condition. The amount administered is sufficient to prevent or inhibit the infection or spread of the infectious agent, such as hepatitis B or HIV, in the recipient. The effect of the compound according to the invention on the viruses mentioned with regard to their prophylactic or therapeutic potential can be seen, for example, as an inhibition of the virus infection, as an inhibition of syncytomy, as an inhibition of the fusion between virus and target membrane, as a reduction or stabilization of the rate of virus replication in an organism or otherwise. The therapeutic effect can consist, for example, in that certain antiviral drugs work better as a desired side effect through the application of the compounds according to the invention, or the number of side effects of these drugs is reduced by reducing the dose. Of course, the therapeutic effect also includes direct action on the viruses in a host. However, this means that the activity of the compounds according to the invention is not limited to eliminating the viruses, but rather encompasses the entire spectrum of advantageous effects in prophylaxis and therapy. Of course, the dose will depend on the age, health and weight of the recipient, the degree of the disease, the type of simultaneous treatment necessary, the frequency of treatment and the type of effects desired and the side effects. The daily Doses of 0.05 to 500 mg / kg body weight can be used once or several times to get the desired results. Daily dose levels are useful in the prevention and treatment of a viral infection including hepatitis B infection. In particular, pharmaceutical agents are typically used for administration approximately 1 to 7 times per day or alternatively or additionally as a continuous infusion. Such administrations can be used as chronic or acute therapy. The amounts of active ingredient that are combined with the carrier materials to produce a single dosage form can of course vary depending on the host to be treated and the particular mode of administration. It is preferred to distribute the target dose over 2 to 5 applications, 1 to 2 tablets with an active substance content of 0.05 to 500 mg / kg body weight being administered for each application. Of course, it is also possible to choose a higher active substance content, for example up to a concentration of up to 5000 mg / kg. For example, you can

Tablets can also be delayed, which reduces the number of applications per day to 1 to 3. The active substance content of the retarded tablets can be 3 to 3000 mg. If, as stated, the active ingredient is administered by injection, it is preferred to bring the host into contact with the compounds according to the invention 1 to 8 times a day or by continuous infusion, amounts of 4 to 4000 mg per day being preferred are. The preferred total amounts per day have proven to be advantageous in human medicine and in veterinary medicine. It may be necessary to deviate from the doses mentioned, depending on the type and body weight of the host to be treated, the type and severity of the disease, the type of preparation for the administration of the medicament and the period or Interval within which the administration takes place. In some cases it may be preferable to bring the organism into contact with less than the stated amounts, while in other cases the specified amount of active ingredient must be exceeded. The person skilled in the art can easily determine the optimum dosages required in each case and the type of application of the active compounds on the basis of his specialist knowledge.

In a further particularly preferred embodiment of the invention, the compounds according to the invention, ie the nucleoside, the nucleic acid, the pharmaceutical agent, the vector, the cells and / or the organism, are administered in a single dose of 1 to 80, in particular 3 to 30 mg / kg body weight used. Like the total amount per day, the amount of the single dose per application can be varied by the person skilled in the art on the basis of his specialist knowledge. The compounds used according to the invention can also be given in the individual concentrations and preparations mentioned together with the feed or with feed preparations or with the drinking water in veterinary medicine. A single dose preferably contains the amount of active ingredient which is administered in one application and which usually corresponds to a whole, half a daily dose or a third or a quarter of a daily dose. The dosage units can accordingly preferably contain 1, 2, 3 or 4 or more single doses or 0.5, 0.3 or 0.25 of a single dose. The daily dose of the compounds according to the invention is preferably divided into 2 to 10 applications, preferably to 2 to 7, particularly preferably to 3 to 5 applications. A permanent infusion of the agents according to the invention is of course also possible. In a particularly preferred embodiment of the invention, 1 to 2 tablets are given for each oral application of the compounds according to the invention. The tablets according to the invention can be provided with coatings and casings known to the person skilled in the art and can also be composed in such a way that they only release the active ingredient (s) in a certain part of the host, if preferred.

In a further preferred embodiment of the invention, the compounds according to the invention can be used with at least one other known pharmaceutical agent. This means that the compounds according to the invention can be used in a prophylactic or therapeutic combination in conjunction with the known medicaments. These combinations can be administered together, for example in a uniform pharmaceutical formulation, or separately, for example in the form of a combination of tablets, injection or other medicaments, which are administered at the same or at different times, with the aim of achieving the desired one to achieve prophylactic or therapeutic effects. These known agents can be agents which increase the action of the nucleosides according to the invention. In particular, it has been found in the antibacterial sector that a wide variety of antibiotics improve the action of nucleosides. This includes agents such as benzylpyrimidines, pyrimidines, sulphoamides, rifampicin, tobramycin, fusidic acid, clindamycin, chloramphenicol and erythromycin. Accordingly, an additional embodiment of the invention relates to a combination in which the second agent is at least one of the aforementioned antiviral or antibacterial agents or classes of agents. It should also be noted that the compounds and Combinations can also be used in conjunction with immunomodulatory treatments and therapies.

There is typically an optimal ratio of the compound (s) according to the invention to one another and / or to other therapeutic or activity-increasing agents (such as transport inhibitors, metabolism inhibitors, inhibitors for renal excretion or glucuronidation, such as sample oath, aceta inophene, aspirin, lorazepan, cimetidine, ranitidine , Colifibrate, indomethacin, ketoprofen, naproxen etc.) in which the active ingredients are present in an optimal ratio. An optimal ratio is defined as the ratio at which the compound (s) according to the invention with another therapeutic agent or agents is such that the overall therapeutic effect is greater than the sum of the effects of the individual therapeutic agents. The optimal ratio is usually found when the means are in the ratio of 10: 1 to 1:10, 20: 1 to 1:20, 100: 1 to 1: 100 and 500: 1 to 1: 500. Occasionally, a negligible amount of a therapeutic agent will suffice to increase the effectiveness of one or more other agents. In addition, the use of the compounds according to the invention in combinations is particularly useful in order to reduce the risk of developing resistance. The compounds according to the invention, such as the nucleosides or nucleic acids, can of course be used in combination with other known anti-viral agents. Such means are known to the person skilled in the art. The compounds according to the invention can accordingly be administered with all conventional agents, in particular other drugs, which are available for use in connection with, in particular, hepatitis drugs, either as individual drugs or in a combination of drugs. They can be administered alone or in combination with them.

It is preferred that the compounds according to the invention are administered with the other known pharmaceutical agents in a ratio of about 0.005 to 1. It is preferred to administer the compounds according to the invention with, in particular, virus-inhibiting agents in a ratio of from 0.05 to about 0.5 part to about 1 part of the known agents. These can also be antitumor or antibacterial agents. The pharmaceutical composition can be in substance or as an aqueous solution together with other materials such as preservatives, buffer substances, agents which are intended to adjust the osmolarity of the solution, etc.

The invention also relates to the use of the nucleic acids according to the invention as anti-sense nucleic acids, in particular in antiviral therapy. The person skilled in the art knows that he can use nucleic acids as anti-sense nucleic acids. The nucleic acid according to the invention preferably serves to prevent the hybridization of the RNA during translation by hybridizing the RNA of the viruses with the nucleic acids according to the invention. The nucleic acids according to the invention can be used in particular as agents against hepatitis B, since they cannot be broken down by the cell's own restriction enzymes or only with difficulty. In general, the nucleic acid according to the invention hybridizes with the DNA of the hepatitis B virus and, in addition to translation, also makes transcription to the virus DNA more difficult.

The nucleosides according to the invention and the nucleic acids according to the invention can be used for the production of pharmaceutical agents. It is thus possible for the teaching according to the invention also to be a method for treating a Viral, bacterial, fungicidal and / or parasitic infection or cancer relates, wherein the nucleosides and / or nucleic acids according to the invention are brought into contact with an organism. The treatment in the sense of the invention includes the prophylactic as well as the therapeutic treatment. The compounds according to the invention can preferably be used to protect organisms, in particular human patients, against a virus infection during a special event, such as childbirth, or for an extended period of time in a country where there is a high risk of infection for hepatitis B. become. The compounds of the invention can then be used alone or with other prophylactic or other antiviral agents that enhance the effectiveness of each agent. Advantageously, the nucleosides according to the invention can be easily adsorbed, preferably after oral administration, into the bloodstream of mammals, in particular human mammals. The compounds advantageously exhibit good water solubility and consistent oral availability. In particular, the good oral availability makes the compounds according to the invention excellent agents for orally administered treatment and prevention courses against a viral infection, in particular a hepatitis B infection. Of course, the compounds according to the invention are not only biologically available orally, but they also advantageously have a high therapeutic index, which above all measures the toxicity against the antiviral effect. Accordingly, the compounds according to the invention are more effective than selected known antiviral agents at lower dose levels and avoid the toxic effect associated with these drugs. The potential of the compound according to the invention to be delivered in doses ^" which far exceed its effective antiviral proportions is particularly evident. partial in slowing down or preventing the possibility of developing resistant variants. Especially during prophylactic treatment, compounds according to the invention can be used in a healthy, but also in a virally infected, in particular infected with a hepatitis B virus, either as a single agent or together with other antiviral agents, which preferably interfere with the replication cycle of hepatitis viruses, be used. The compounds according to the invention are used in prophylaxis and therapy according to the routes known to the person skilled in the art. If the method for treating a viral infection with the nucleosides according to the invention is a combination therapy, each agent used, that is to say the known compounds and the compound according to the invention, has an additive, non-additive or synergistic effect in inhibiting virus replication, since it can advantageously be provided that each agent acts at a different point in the replication of the viruses. The method of such combination therapies can also advantageously prevent the dosage of a conventional antiviral agent for which a desired therapeutic or prophylactic effect would be required compared (when the agent is administered alone). Such combinations of the method according to the invention for the treatment of viral diseases can reduce or eliminate the side effects of conventional therapies with individual antiviral agents, wherein they advantageously do not interfere with the antiviral effect of these agents, but rather increase synergistically. These combinations reduce the potential for resistance to single agent therapy while advantageously minimizing the associated toxicity. These combinations can also increase the effectiveness of the conventional agent without increasing the associated toxicity. In particular, it is preferred that the compounds according to this invention, together with other antiviral or antibacterial or fungicidal agents, in an additive or synergistic manner prevent the replication of the genetic material of viruses. Preferred combination therapies include administration of a compound of the invention with ACTddi, ddC, d4T, 3TC, or a combination thereof. It is of course also possible that in the method according to the invention or when using the compounds according to the invention, administration with other nucleoside derivatives or with viral reverse transcriptase inhibitors or protease inhibitors is preferred. Co-administration of the compounds of the invention with reverse viral transcriptase or aspartyl protease inhibitors shows an additive or synergistic effect whereby the virus replication or infection or both or both symptoms associated therewith are prevented, substantially reduced or completely eliminated become. The administration of a combination of agents may be preferred over the administration of individual agents. The compounds according to the invention can also be used together with immunomodulators or immunostimulators; preferred immunomodulators or immunostimulators are: propirimine, anti-human α-interferon antibodies, IL-2, GM-CSF, interferon α, diethyldithiocarbamate, tumor necrosis factor, naltrexone, Tuscarasol, rEPO and antibiotics such as pentamidine isethionate; but also agents that prevent or fight malignant tumors associated with viral diseases. In the process for the treatment of viral, bacterial, fungicidal and / or parasitic infection or cancer, the compounds according to the invention - as already explained above - can be administered with compatible carriers, adjuvants or vehicles. Pharmaceutically acceptable carriers, adjuvants and ve Products that can be used in the medicaments of this invention include ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as dα-tocopherol polyethylene glycol 1000 succinate, or other similar polymeric delivery matrices, serum proreins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acids, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogenphosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisiliconosulfonate, polyvinyl trisiliconosulfonate, polyvinyl sulfonate, polyvinylsulfonate , Polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fat, but are not limited thereto. Cyclodextrins, such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives, such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilized derivatives can also be used advantageously, to increase the delivery of the compounds of the invention. In connection with the method, the compounds according to the invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Oral administration or administration by injection is preferred as the form of contacting. The pharmaceutical compositions of this invention can contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, the pH of the formulation can be adjusted with pharmaceutically acceptable acids, bases or buffers to increase the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intra- muscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion procedures in the form of contacting.

The invention also relates to a kit which comprises the compounds according to the invention, optionally with information about combining the contents of the kit. The information on combining the contents of the kit relates to the use of the kit for the prophylaxis and / or therapy of diseases, in particular viral diseases. The information can also relate, for example, to a therapy scheme, ie to a specific injection or application scheme, to the dose to be administered or something else.

The nucleoside analogs according to the invention have numerous advantages. Human and animal organisms have to deal with numerous pathogens in the course of their individual development. These pathogens can represent fungi, bacteria, but especially viruses. Every year, millions of people and farm animals worldwide contract viral diseases, with numerous of these infections being associated with significant health impairments. Diseases with the human immunodeficiency virus with the hepatitis viruses or HIV can sometimes lead to death if left untreated.

The viruses that organisms have to deal with differ greatly from their infectious potential. The very infectious viruses include the hepatitis B viruses (HBV), which can cause inflammation of the liver, which is regularly accompanied by damage to the liver cells. ten viruses, such as the hepatitis viruses B, C and D, which can develop liver damage up to the liver tumor.

Various antiviral therapies have been developed in the prior art in order to be able to successfully fight viruses in a host organism - for example a human being or an agricultural product or pet. Many of these therapies are chemotherapies that are intended to prevent the replication of pathogenic viruses in the host cell. Various phases of replication such as adsorption, penetration, translation, transcription of the viral genes, replication of the nucleic acid and the assembly of the virus particles can be used as a point of attack for the so-called virus statics used here. The virus adsorption inhibitors interact with cationic regions of the viral coat protein and thereby prevent association with the receptors of the potential host cell. In contrast to the adsorption inhibitors, the inhibitors of virus-cell fusion do not already prevent the binding, but only the fusion with the host cell with the formation of a common membrane. Another possibility is the inhibition of the penetration with release of the viral genome, as described in the prior art for Picorna viruses, for example. It is also possible to block the transcription and protein biosynthesis of the viruses. The prior art also describes methods for inhibiting viral DNA polymerase. The inhibition of viral DNA polymerase is disclosed in the prior art, in particular for herpes viruses. The DNA

Herpesvirus polymerase has different functions. Among other things, it is responsible for introducing the viral genetic information of the genome of the host cell, for RNA-dependent DNA synthesis, for DNA-dependent DNA synthesis and has other tasks. Many of the Antiviral compounds successfully used today are nucleoside-analogous substances. however, their antiviral activity is particularly limited to herpes viruses.

Since the strategies mentioned are particularly successful with herpes viruses and can sometimes be used less successfully with other viruses, it was necessary to develop different therapies for each different virus group. For example, genetically engineered vaccines have been available for the treatment of hepatitis B for years, but they can no longer help infected people and can no longer significantly influence the chronic course of this disease mentioned. The nucleosides according to the invention avoid these listed disadvantages of the prior art.

The invention is to be explained in more detail below with the aid of examples, without being restricted to these examples.

Examples

1. Synthesis of ß-L-5-methyldeoxycytidine (ß-L-MetCdR)

1.1 1- (3,5-Di-O-acetyl-2-deoxy-β-L-ribofuranosyl) thymine.

L-thymidine (7.65 mmol, 1.85 g) is dissolved in dry pyridine (15 ml). The mixture is washed with acetic anhydride (2.9 ml,

30.6 mol) are added and remain with stirring for 12 hours at room temperature. Then the solvent in Vacuum removed. The residue is dissolved in chloroform, the solution obtained is washed twice with saturated sodium bicarbonate solution and then with water. The organic phase is dried with sodium sulfate. After the solvent has been driven off in vacuo, the residue obtained is purified by column chromatography on silica gel with chloroform / methanol (99/1, v / v) as the eluent. 2.4 g of the di-O-acetyl compound are obtained as a white foam from the corresponding fractions.

1.2 1- (2-Deoxy-ß-L-ribofiuranosyl) -5-methylcytosine (ß-L-5-methyldeoxycytidine, ß-L-MetCdR).

1- (3, 5-Di-0-acetyl-2-deoxy-ß-L-ribofuranosyl) thymine (2.4 g, 7.36 mmol) is dissolved in anhydrous pyridine (50 ml), with 1H-1, 2, 4-Triazole (1.02 g, 11.14 mmol) and chlorophenyl dichlorophosphate (1.8 ml, 11.14 mmol) were added. The reaction mixture remains at room temperature with stirring for four days. The pyridine is then removed in vacuo, the residue is dissolved in dioxane / 25% NH ₄ OH (3/1, v / v) and stored overnight at room temperature. The solvent is removed in vacuo, the dark residue obtained is dissolved in water (40 ml) and placed on a column (2 × 20 cm) which contains DOWEX 50 WX 8 (H ⁺ form). It is eluted first with water (500 ml) and then with 5% ammonia solution (800 ml). The ammoniacal eluate is evaporated to dryness in vacuo. The residue is purified by chromatography on silica gel with the upper phase of the solvent mixture ethyl acetate / i-propanol / water (4/1/2, v / v). The target connection, ß-L- Methyl deoxycytidine is obtained as a yellowish foam (1.3 g), from which 200 mg are branched off, dissolved in a little methanol and a few drops of methanolic HCl are added. The crystals which have separated out after some time are filtered off. The hydrochloride of ß-L-methyldeoxycytidine thus obtained has a melting point of 151-153 ° C (dec.).

UV spectrum (H ₂ 0, pH 7) λ _max 281 n (ε 11644), λ _min 251 nm (ε 5448); (H ₂ 0, pH 1) λ _max 287 nm (ε 14770), λ _min 245 nm (ε 2238).

^ -NMR (Me ₂ SO-d ₆ ) δ (ppm) = 1.85 (s, 3H, CH ₃ ), 1.92-1.97 (m, 1H, H-2 "), ^' 2.04-2.07 (m, 1H, H -2 ""), 3.42 - 3.57 (m, 2H, H-5 "and H-5""), 3.74 (, 1H, H-4"), 4.20 (m, 1H, H-3 "), 5.07 (br d, 1H, OH-3 "), 5.21 (br t, 1H, OH-5"), 6.15 - 6.17 (dd, 1H, Hl "), 6.77 (br s, 1H, NH), 7.26 (br s, 1H, NH), 7.61 (S, 1H, H-6).

¹³ C-NMR (Me ₂ S0) δ (ppm) = 13.3 (CH ₃ ), 40.2 (C-2 "), 61.4 (C- 5"), 70.4 (C-3 "), 84.6 (Cl"), 87.1 (C-4 "), 101.27 (C-5), 138.30 (C-6), 155.20 (C-2), 165.31 (C-4). Analysis calculated for Cι ₀ Hι _β N ₃ O ₄ Cl: C, 43.25; H, 5.81; N, 15.13. Found: C, 43.11; H, 5.97; N, 14.89.

2. Synthesis of ß-L-2 ", 3 -Didehydro-2", 3 "-dideoxy-5-ethylcytidine (ß-L-ddeMetC)

2.1 (2-deoxy-ß-L-ribofuranosyl) -N-benzoyl-5-methylc tosin.

ß-L-5-methyldeoxycytidine (7.25 g, 30 mmol) is dissolved under an argon protective atmosphere in anhydrous dimethylformamide (200 ml) and treated with benzoic anhydride (7.5 g, 33 mmol) added. The reaction mixture is stirred for 24 hours at room temperature. The solvent is then removed in vacuo. The resulting residue is triturated with ether, filtered off, washed with ether and dried. 9.3 g of 1- (2-deoxy-ß-L-ribofuranosyl) - N-benzoyl-5-methylcytosine are obtained as a crude product.

2.2 1- (2-deoxy-3,5-di-O-mesyl-β-L-ribofuranosyl) -N-benzoyl-5-methylc tosi.

To a solution of the above-described N-benzoyl derivative (2.2 g, 6.4 mmol) in pyridine (20 mL) at - (1.0 ml, 13.6 mmol) ^• 5 ° C, methanesulfonyl chloride is added. After 18 hours the solution is poured into an ice / water mixture (200 ml) with vigorous stirring. The precipitated product is air dried. Yield: 2.9 g.

2.3 1- (2-deoxy-3,5-epoxy-β-L-threopentofuranosyl) -5-methylcytosine.

A solution of the di-O-mesyl compound described under 2.2 (2.26 g, 4.52 mmol) in ethanol (150 ml) and water (35 ml) containing sodium hydroxide (15 ml of an IN solution) is added for two hours heated under reflux. After cooling, the solution is neutralized with dilute acetic acid and concentrated to dryness. The residue is extracted several times with hot acetone (5 x 50 ml). The acetone solution is concentrated and the residue obtained is suspended in cold ethanol (7 ml). The precipitated crystals are filtered off (0.55 g). 2.4 1- (2,3-dideoxy-ß-L-glyceropent-2-enofuranosyl) -5-ethylcytosine (2 ", 3" didehydro-2 ^* , 3 "-dideoxy-5-methylcytidine (ß-L- ddeMetC).

The 3 ', 5' epoxy derivative of β-L-5-methylcytidine shown in 2.3 becomes a solution of potassium tert-butoxide (1.93 g, 17 mmol) in 55 ml of anhydrous dimethyl sulfoxide

(1.75 g, 8.3 mmol) and added for 3 hours

Room temperature stirred. The solvent is removed in vacuo, the residue dissolved in water (7.5 ml) and neutralized with acetic acid / methanol (l / l, v / v). The neutral solution is evaporated to dryness, the residue is separated by column chromatography on silica gel with chloroform / methanol (85/15, v / v) as the eluent. From the. appropriate fractions are obtained after the solvent has been driven off ß-L-ddeMetC (0.72 g). UV spectrum (H ₂ 0, pH 7) γ _max 278 nm (ε 10067), γ _min 253 (ε 5668); (H ₂ 0, pH 1) γ _max 285 nm (ε 9240), γ _{m ± n} 244 nm (ε 1628). ^ Η NMR (Me ₂ SO-d ₆ ): δ (ppm) = 1.79 (s, 3H, CH ₃ ), 3.57 (m, 2H, H-5 "and H" -5 ""), 4.74 (m , 1H, H-4 "), 4.97 (t, 1H, OH-5"), 5.86 (, 1H, H-2 "), 6.31 (m, 1H, H-3"), 6.79 (br s, 1H , NH) 6.89 (m, 1H, Hl "), 7.28 (br s, 1H, NH), 7.53 (s, 1H, H-6). ¹³ C-NMR (Me ₂ SO-d ₆ ): δ (ppm) = 13.26 (CH ₃ ), 62.59 (C-2 "), 86.99 (C-5"), 89.56 (C-3 "), 101.18 (Cl"), 126.92 (C-4 "), 133.89 ( C-5), 138.84 (C-6), 155.40 (C-2), 165.43 (C-4).

Analysis calculated for C ₁₀ H ₁₃ N ₃ O _3: C, 53.81; H, 5.87; N, 18.82. Found: C, 53.57; H, 5.76; N, 18.86.

3. Synthesis of 1- (2,3-dideoxy-ß-L-glyceropentofuranosyl) -5-methylcytosine (ß-L-2 ", 3" - dideoxy-5-methylcytidine (ß-L-ddMetC) 1- (2, 3-Dideoxy-β-L-glyceropent-2-enofuranosyl) -5-methylcytosine (0.22 g, 1 mmol) is dissolved in methanol (80 ml). Palladium / carbon (10%) is added and hydrogenolyzed at room temperature. The thin layer chromatographic control shows no starting product after 30 minutes. The catalyst is filtered off, the filtrate is concentrated, and the resulting residue is chromatographed on a silica gel column using chloroform / methanol (9/1, v / v) as the eluent. The corresponding fractions are pooled. After removal of the solvent, a residue is obtained which, after crystallization from methanol, gives pure 1- (2,3-dideoxy-β-L-glyceropentofuranosyl) -5-methylcytosine (L-ddMetC, 103 mg).

^X H NMR (Me ₂ SO-d ₆ ): δ (ppm) = 1.67 (d, 3H, CH ₃ ), 2.37 - 2.63 (m, 4H, H-2 ", H-2"", H-3 "and H-3""), 3.78 (m, 2H, H-5" and

H-5 ""), 4.08 (m, 1H, H-4 "), 5.10 (br t, 1H, 5" -OH), 6.85 (dd, 1H, Hl "), 7.12 (br s, 1H, NH ), 7.23 (br s, 1H, NH), 7.64 (s, 1H, H6).

¹³ C-NMR (Me ₂ SO-d ₆ ): δ (ppm) = 13.7 (CH ₃ ), 38.3 (C-2 "), 65.4 (C-5"), 69.1 (C-3 "), 81.3 ( Cl "), 86.4 (C-4"), 116.2 (C-5), 137.7 (C-6), 153.5 (C-2), 164.7 (C-4). Analysis calculated for Cι ₀ Hι ₅ N ₃ O ₃ : C, 53.31; N 6.72; N, 18.66. Found: C, 53.43; H, 6.70; N, 18.73.

4. Synthesis of l-ß-L-arabinofuranosyl-5-methylcytosine (ß-L-AraMetC)

1-ß-L-Ribofuranosyl-thymine is converted into the 5 -O-trityl ether in the usual way with triphenylchloromethane. This compound 5.4 g (10.8 mmol) is suspended in toluene (120 ml), warmed to 80 ° C. and with a solution of N, N "-thiocarbonyldiimidazole (2.16 g, 12.1 mmol) in toluene (80 ml). The mixture is refluxed for two hours. After cooling, the solid material is filtered off and recrystallized from ethanol. 4.3 g of 2.2 "anhydro-5" -0-trityl- L-ribofuranosylthymine The anhydro compound (4.3 g, 10 mmol) is placed in 50% ethanol (400 ml), 1 N NaOH (25.5 ml) is added and the suspension is heated to 60 ° for two hours C. After cooling, the solution is neutralized with acetic acid. The ethanol is distilled off, 1- (5 "-0-trityl-ß-L-arabinofuranosyl) thymine precipitates out of the remaining aqueous portion. 4.4 g of this compound are obtained, which is dissolved in methanol (250 ml) and concentrated hydrochloric acid (25 ml) is added. After two hours the 5 "-0-trityl group has been split off completely. After the solvent has been driven off, the resulting residue is separated by column chromatography on silica gel using a chloroform / methanol mixture (8/2) as the eluent. From the corresponding

Fractions are 1.7 g (6, 6 mmol) 1-ß-L-

Arabinofuranosylthymine obtained. The conversion of this

Connection into the 1-β-L-arabinofuranosyl-5-methyl-cytosine takes place in an analogous manner as described under Examples 1.1 and 1.2.

The melting point of the hydrochloride is 201-203 ° C (dec.). UV spectrum (H ₂ 0, pH 7) λ _max 281 nm (ε 11796), λ _min 250 nm (ε 4600); (H ₂ 0, pH 1) λ _ma χ 288 nm (εl4633), λ _mn 247 nm (ε3800). ^X H-NMR (Me ₂ SO-d ₆ ): δ (ppm) = 1.84 (s, 3H, CH ₃ ), 3.60 (m, 2H, H-5 "and H-5""), 3.71 (d, 1H, H-4 "), 3.88 (m, 1H, H-3"), 3.94 (m, 1H, H-2 "), 5.05 (s, 1H, 5" -OH), 5.34 (s, 1H, 3'- OH), 5.38 (s, 1H, 2 '-OH), 6.19 (d, 1H, Hl "), 6.71 (br s, 1H, NH), 7.20 (br s, 1H, NH), 7.42 ( s, 1H, H-6).

¹³ C-NMR (Me ₂ SO-d ₆ ): δ (ppm) = 13.3 (CH ₃ ), 61.1 (C-5 "), 74.9 (C-3"), 76.2 (C-2 "), 84.6 ( C-4 "), 85.5 (Cl"), 99.2 (C-5), 140.2 (C-6), 155.2 (C-2), 165.2 (C-4).

Analysis calc. For Cι ₀ Hι ₅ N ₃ O ₅ : C, 46.69; H, 5.88; N, 16.33. Found: C, 46.81; H, 5.64; N, 16.37.

5. Synthesis of l- (3 "-azido, 2", 3 "-dideoxy- - ribofuranosyl) -5-methyl c tosin), ß-L-3" -azido-2 ", 3" - dideoxy- 5 - methylcytidine (ß-LN ₃ MetCdR)

1- (3'-Azido-2 ', 3' -dideoxy-ß-L-ribofuranosyl) thymine (3.1 g; 11.6 mmol) is dissolved in pyridine (50 ml) and cooled with acetic anhydride (2 , 8 ml; 30 mmol) was added. The mixture remains at room temperature with stirring for 12 hours. The solvent is then removed in vacuo and the residue is dissolved in chloroform. The solution is washed twice with saturated sodium bicarbonate solution and with water. The organic phase is dried with sodium sulfate, the solvent is removed in vacuo and the resulting residue is purified by column chromatography on silica gel with chloroform / methanol (97/3, v / v) as the eluent. 2.9 g of 1- (5 "-0-acetyl-3" -azido-2 ", 3" -dideoxy-ß-L-ribofuranosyl) thymine are obtained as foam from the corresponding fractions (yield: 81%). This compound is dissolved in pyridine (50 ml) with 1H-1, 2, 4-triazole (1.37 g; 15 mmol) and chlorophenyl dichlorophosphate (2.42 ml; 15 mmol) were added. The mixture remains at room temperature for four days. The pyridine is then removed in vacuo, the residue is dissolved in dioxane / 25% ammonia (50 ml; 3/1; v / v) and stored overnight at room temperature. The solvent is removed in vacuo, the residue obtained is dissolved in water (50 ml) and placed on a column (2 × 20 cm) which contains DOWEX 50 WX 8 (H ⁺ form). It is eluted first with water (600 ml), then with 5% aqueous ammonia solution (750 ml). The ammonia-alkaline eluate is evaporated to dryness and the residue is purified by chromatography on silica gel with chloroform / methanol (9/1; v / v) as the mobile phase. 1- (3 "-Azido-2", 3 "-dideoxy-ß-L-ribofuranosyl) -5-methylcytidine is isolated from the corresponding fractions as a yellowish amorphous product (1.1 g; 41.7%).

^X H-NMR (Me ₂ SO): δ (ppm) = 1.78 (s, 3H, CH ₃ ), 1.98 - 2.09 (m, 2H, H-2 "and H-2""), 3.56 - 3.63 (m , 2H, H-5 "and H-5""), 3.86 (m, 1H, H-4"), 4.12 (m, 1H, H-3 "), 5.17 (br t, 1H, 5" - OH ), 6.15 - 6.18 (dd, 1H, Hl "), 6.83 (br s, 1H, NH), 7.56 (br s, 1H, NH), 7.86 (s, 1H, H-6).

¹³ C-NMR (Me ₂ S0-d ₆ ): δ (ppm) = 20.2 (CH ₃ ), 36.7 (C-2 "), 60.8 (C-5"), 75.3 (C-3 "), 84.7 ( Cl "), 88.1 (C-4"), 108.2 (C-5), 140.5 (C-6), 152.7 (C-2), 164.3 (C-4).

6. Determination of the antiviral activity of β-L-5-methylcytosine nucleosides

The antiviral activity of the compounds according to the invention was investigated on HepG2 2.2.15 cells, a human hepatoblastoma cell line which the contains replication-competent HBV genome stably integrated and productively produces infectious progeny viruses (Seils et al., Proc Natl Acad Sei USA 1987, 84: 1005-1009). These cells were cultivated under the standardized conditions specified by Korba and Gerin and the amount of extracellular viral DNA was determined (Korba et al. Antiviral Res 1992, 19: 55-70).

HepG2 2.2.15 cells were seeded with a density of about 60% in 12-well plates and cultured in 10% FBS Dulbecco MEM to confluence. The medium was then switched to 2% FBS and the cells were cultured for a further 24 h.

After changing the medium again, the cells were treated with different concentrations of the compounds according to the invention. The connections were also added to the medium every 24 hours. On the 6th day of treatment, the cell supernatants were centrifuged off and stored at -20 ° C. until analysis of the HBV DNA. After treatment of the culture supernatants with proteinase K, the extracellular viral DNA was amplified by PCR using the following primers (forward: 5 "-CTC CAG TTC AGG AAC AGT AAA CCC-3"; reverse: 5 "-TTG TGA GCT CAG AAA GGC CTT GTA AGT TGG CG-3 ". The PCR products were separated on 1% agarose, stained with ethidium bromide and quantified with the Fluor-S ™ Multimager (Biorad).

Serial dilutions of the pUC19 HBV and pTHBV plasmids with known genome equivalences (GE) were used for the calibration of the PCR reaction. This resulted in a lower detection limit of approximately 10 ³ units and a linearity between 10 ³ and 10 ⁵ units. Table 1 shows the concentrations of the compounds according to the invention which are required after a 9-day incubation of the HepG2 2.2.15 cells for a 50% reduction in the extracellular HBV-DNA (ED ₅₀ ).

Table 1. The effectiveness of the β-L-5-methylcytidine nucleosides compared to 3TC (lamivudine) on HBV replication in HepG2 2.2.15 cells. The concentrations are shown which lead to a 50% reduction in HBV DNA in the medium of the cells (ED ₅₀ values).

ED ₅₀ values; uM

Inhibition of HBV DNA polymerase by β-L-5-methylcytidine nucleoside triphosphates The synthesis and purification of the triphosphates of the β-L-5-methylcytidine nucleosides was carried out according to known methods (Yoshikawa et al., Tetradedron Lett 1967, 50: 5065-5068 ; Hoard et Ott, J Am Chem Soc 1965, 87: 1785-1788). For the determination of the endogenous HBV DNA polymerase activity, about 60 ml of serum from patients with hepatitis B infections from Charite, Berlin (> 10 ⁷ HBV Particles / ml) centrifuged at 3000 rpm, then the virus particles at 25,000 g, sedimented for 60 min, in 7 ml of TKM buffer (50 mM Tris-HCl, pH7, 5, 50 mM KC1, 5 mM MgCl ₂ ) and taken up by a Sucrose gradients (0.3 M, 0.6 M, 0.9 M sucrose in 10 ml TKM buffer)

25,000 g, centrifuged for 20 hours. The cleaned virus sediment was suspended with ultrasound in 400 ul TKM, aliquoted and frozen at -80 ° C (Davies et al., Antiviral Res 1996, 30: 133-145). The polymerase mixture contained in 30 μl about 2-4 × 10 ^{8 of} the purified virus particles (which had previously been lysed in 6% β-mercaptoethanol, 10% Igepal for 15 min at room temperature), 42 mM Tris-HCl (pH 7.5) and 34 mM MgCl ₂ , 340 mM KC1, 22 mM β-mercaptoethanol, 0.4% Igepal, 70 μM TTP, dATP, dGTP and 1 μCi ³ H-dCTP (= 0.7 μM dCTP) (Matthes et al., Antimicrob Agents & Chemoth 1991 , 35: 1254-1257) and various concentrations of the β-L-5-methylcytosine nucleoside triphosphates. After two hours of incubation at 37 ° C., 20 μl of the batches were placed on paper filters, washed 5 times with 5% trichloroacetic acid and 0.1% Na pyrophosphate, and the ³ H-dCMP incorporated into the HBV DNA was then measured in a liquid scintillation counter. The concentration-dependent β-L-5-methylcytidine nucleoside triphosphates, which lead to a 50% inhibition of HBV DNA polymerase activity, were determined from the concentration-dependent inhibition curves of the HBV DNA synthesis. These IC ₅₀ values are shown in Table 2. Table 2. Inhibition of HBV DNA polymerase by ß-L-5-methylcytidine nucleoside triphosphates compared to 3TC triphosphate (IC ₅₀ values).

Cytotoxicity of ß-L-5-methylcytosine nucleosides

For this purpose, established cells of human myeloid leukemia (HL-60) in RPMI medium, or the already mentioned HepG2 cells in Dulbecco-MEM, were incubated for two days with different concentrations of the compounds and then the proliferation rate of the cells was determined. The concentration of the compounds, which leads to a 50% inhibition of cell proliferation, was determined from the data (CD ₅₀ ). Table 3. Cytotoxicity (CD ₅₀ ) of β-L-5 methylcytidine nucleosides towards HL-60 and HepG2 cells.

Claims

claims

1. New β-L-5-methylcytosine nucleosides of the formulas I and II for the treatment and prophylaxis of hepatitis B infections

Formula I.

where Rx = H, OH, FR ₂ = H, OH R ₃ = HR ₄ = H, OH, N ₃ and R ₅ = H, acetyl, palmitoyl, alkoxycarbonyl, carbamate, phosphonate, monophosphate, diphosphate or triphosphate , if R _{1 #} R ₂ and R ₃ = H, then R ₄ = H, OH or N ₃ , -when R = OH or F, then R ₂ and R ₃ = H and R ₄ = OH,

Formula II

where R ₆ = H, F and R ₇ = H. β-L-nucleoside according to claim 1, characterized in that it is β-L-5-methyldeoxycytidine,

3. ß-L-nucleoside according to claim 1, characterized in that it is ß-L- 2 ", 3" -dideoxy-5-methylcytidine. β-L-nucleoside according to claim 1, characterized in that it is β-L-arabinofuranosyl-5-methylcytosine

5. β-L-nucleoside according to claim 1, characterized in that it is β-L- 2 "-fluoroarabinofuranosyl-5-methylcytosine.

6. β-L-nucleoside according to claim 1, characterized in that it is β-L-3 "-azido-2", 3 "-dideoxy-5-methylcytidine.

7. β-L-nucleoside according to claim 1, characterized in that it is β-L-2 ", 3" -didehydro-2 ", 3" -dideoxy-5-methylcytidine.

8. β-L-nucleoside according to claim 1, characterized in that it is β-L-2 ", 3" -didehydro-2 ", 3" -dideoxy-2 "-fluoro-5-methylcytidine.

9. β-L-nucleoside according to one of the preceding claims, the group comprising a salt, a phosphonate, a monophosphate, diphosphate, triphosphate, another ester or a salt of such esters.

10. β-L-nucleoside according to one of the preceding claims, for use as a medicament.

11. Nucleic acids comprising as a building block at least one β-L nucleoside according to claims 1 to 9.

12. Pharmaceutical composition comprising a β-L-nucleoside or a derivative according to one of claims 1 to 9 and / or a nucleic acid according to claim 11 optionally together with customary auxiliaries, preferably carriers, adjuvants and / or vehicles.

13. Pharmaceutical agent according to the preceding claim, characterized in that it further comprises one or more additional agents from the group of antiviral, fungicidal or antibacterial agents, anti-cancer agents and / or immunostimulators or immunomodulators.

14. Pharmaceutical agent according to the preceding claim, characterized in that the antiviral agents protease inhibitors and / or reverse transcriptase inhibitors and / or HBV DNA polymerase inhibitors, immunostimulators bropirimine, anti-human alpha-interferon antibodies, IL- 2, GM-CSF, interferons, diethyldithiocarbamate, tumor necrosis factors, naltrexone, tuscarasol and / or rEPO.

15. Pharmaceutical agent according to the preceding claim, characterized in that it contains one or more additional anti-HBV active agents from the group comprising PMEA (adefovir dipivoxil), famciclovir, penciclovir, diaminopurine dioxolane (DAPD), clevudine (L-FMAU ), Entecavir, interferon or thymosin α 1 and / or inhibitors of nucleocapsid formation, in particular heteroaryl pyrimidines.

16. Pharmaceutical agent according to one of the preceding claims, characterized in that the agents are pegylated.

17. Pharmaceutical agent according to the preceding claim, characterized in that it contains one or more additional agents which are able to switch off the function of cellular proteins which are essential for the multiplication of HBV.

18. Pharmaceutical composition according to the preceding claim, characterized in that it is effective against hepatitis B viruses which are resistant to lamivudine or another cytosine nucleoside, such as. B. Emtricitabine (L-FTC), L-ddC or L-ddeC.

19. Pharmaceutical agent according to the preceding claim, characterized in that it prevents cancer.

20. Pharmaceutical composition according to the preceding claim, characterized in that it prevents the formation of a liver cancer as a result of chronic hepatitis triggered by the HBV.

21. Pharmaceutical composition according to one of the preceding claims, characterized in that the carriers are selected from the group comprising fillers, extenders, binders, humectants, disintegrants, solution retarders, absorption accelerators, wetting agents, adsorbents and / or lubricants.

22. Use of the β-L-nucleosides according to one of claims 1 to 9, a nucleic acid according to claim 11 and / or a pharmaceutical agent according to one of claims 12 to 21 in the prophylaxis or therapy of a viral, bacterial, fungicidal and / or parasitic Infection or cancer.

23. Use according to the preceding claim, characterized in that the viral disease is associated with a hepatitis virus, an HIV, a bovine immunodeficiency virus, a capric arthritis encephalitis virus, an equine infectious anemia virus, an ovine Maedi-Visna virus, a Visna lentivirus , an avian leukemia virus, a human T-cell leukemia virus and / or a feline immunodeficiency virus.

24. Use according to the preceding claim, characterized in that the hepatitis virus is a hepatitis B or hepatitis D virus.

25. Use according to the preceding claim, characterized in that HIV is HIV-0, HIV-1 and / or HIV-2.

26. Use according to one of the preceding claims, characterized in that the ß-L nucleoside according to one of claims 1 to 9, the nucleic acid according to claim 11 and / or the pharmaceutical agent according to one of claims 12 to 21 as a prodrug, as Feed and / or as a drinking water additive.

27. Use according to one of the preceding claims, characterized in that it is in the form of a gel, powder, powder, tablet, slow-release tablet, premix, emulsion, infusion formulation, drop, concentrate, granulate, syrup, pellet, bolus, capsule, aerosol, spray and / or inhalate is prepared and / or applied.

28. Use according to one of the preceding claims, characterized in that the ß-L nucleoside according to one of claims 1 to 9, the nucleic acid according to claim 11 and / or the pharmaceutical agent according to one of claims 12 to 21 in a concentration of 0 , 1 to 99.5, preferably from 0.5 to 95, particularly preferably from 20 to 80% by weight is present in a preparation.

29. Use according to one of the preceding claims, characterized in that the ß-L nucleoside according to one of claims 1 to 9, the nucleic acid according to claim 11 and / or Pharmaceutical composition according to one of claims 12 to 21 is used orally, rectally, subcutaneously, intravenously, intramuscularly, intraperitoneally and / or topically.

30. Use according to one of the preceding claims, characterized in that the β-L-nucleoside according to one of claims 1 to 9, the nucleic acid according to claim 11 and / or the pharmaceutical agent according to one of claims 12 to 21 in total amounts of 0, 05 to 500 mg / kg, preferably from 5 to 100 mg / kg body weight is used per 24 hours.

31. Use according to one of the preceding claims, characterized in that the ß-L nucleoside and / or the nucleic acid is used in a single dose of 1 to 80, in particular 3 to 30 mg / kg body weight.

32. Use according to one of the preceding claims, characterized in that the β-L nucleoside according to one of claims 1 to 9, the nucleic acid according to claim 11 and / or the pharmaceutical agent according to one of claims 12 to 21 to 2 to 10 daily Applications is distributed, preferably 3 to 5.

33. Use according to the preceding claim, characterized in that 1 to 2 tablets are given for each oral application.

34. Use according to any one of the preceding claims, characterized in that the ß-L nucleoside according to one of claims 1 to 9, the nucleic acid according to claim 11 and / or the pharmaceutical agent according to one of claims 12 to 21 in combination with at least one other known pharmaceutical agents is used.

35. Use according to the preceding claim, characterized in that the ß-L nucleoside according to one of claims 1 to 9, the nucleic acid according to claim 11 and / or the pharmaceutical agent according to one of claims 12 to 21 have a therapeutic effect of the other known strengthen pharmaceutical agents non-additively, additively or synergistically, increase the therapeutic index and / or reduce the risk of toxicity from the respective compound.

36. Use according to the preceding claim, characterized in that the ß-L nucleoside according to one of claims 1 to 9, the nucleic acid according to claim 11 and / or the pharmaceutical agent according to one of claims 12 to 21 with the other known pharmaceutical agents be administered in a ratio of about 0.005 to 1.

37. Use according to one of the preceding claims, characterized in that the, ß-L-nucleoside according to one of claims 1 to 9 is used in combination with 3-deazauridine.

38. Use of the β-L-nucleosides according to one of claims 1 to 9 and / or the nucleic acid according to claim 11 for the production of pharmaceutical compositions.

39. A method for treating a viral, bacterial, fungicidal and / or parasitic infection or cancer, characterized in that the ß-L-nucleoside according to any one of claims 1 to 10, the nucleic acid according to claim 11 and / or the pharmaceutical agent according to one of claims 12 to 21 are brought into contact with an organism.

40. Kit comprising the β-L-nucleoside according to one of claims 1 to 10, the nucleic acid according to claim 11 and / or the pharmaceutical agent according to one of claims 12 to 21, optionally with information for combining the contents of the kit.

41. Use of the kit according to the preceding claim for the prophylaxis or therapy of viral diseases.