DE19962010A1 - Antiviral combination based on dihydropyrimidine derivatives and non-dihydropyrimidine HBV antiviral agents, are useful for treatment of HBV infections - Google Patents

Abstract

A synergistic combination comprising dihydropyrimidines and HBV antiviral agents other than dihydropyrimidines, is new. A synergistic combination comprises: (A) at least one dihydropyrimidine; (B) at least one HBV antiviral agent other than (A); and optionally (C) at least one immunomodulator. An Independent claim is also included for a combination of HBV core protein inhibitor(s), HBV antiviral agent(s) other than HBV core protein inhibitors and optionally immunomodulator(s).

Description

The invention relates to combinations of non-nucleoside inhibitors, such as z. Dihydropyrimidines, and other antiviral agents, a method of theirs Production and its use as a medicament, in particular for treatment and prophylaxis of HBV infections.

"Combinations" within the meaning of the invention are not just Darreichungs forms containing all components (so-called fixed combinations), and Kombina tion packages containing the components separated from each other, but also simultaneously or temporally staggered applied components, if they used for the treatment or prophylaxis of the same disease.

The hepatitis B virus belongs to the family of hepadna viruses. It causes a acute and / or persistent progressive disease. diverse other clinical manifestations in the clinical picture are caused by the hepatitis B virus Virus causes - especially chronic liver inflammation, liver cirrhosis and hepatocellular carcinoma. Furthermore, coinfection with the hepatitis Delta virus negatively affect the disease process.

Several options have already been proposed for virus inhibition:
the inhibition of the polymerase of HBV by analogs of the substrates of this enzyme such as lamivudine, FTC, adefovir dipivoxil, abacavir, β-L-FDDC, L-FMAU and BMS 200 475:

The polymerase is one of the essential and essential in the multiplication cycle enzymatic activities of the HBV virus; see. Radziwill et al., J. Virol. 64, 613- 620 (1990). Analogues of natural substrates - mostly after phosphorylation through the host 's own enzymes - the viral polymerase complex inhibit the  Polymerase activity and thus the replication of HBV in vitro and in vivo; see. z. Chang et al., J. Biol. Chem. 267, 13938-13942 (1992).

Lamivudine has recently been used to treat chronic HBV-infected people Patients used. However, it shows a high after discontinuation of therapy Rebound effect and leads to resistance during long-term therapy. For adefovir Dipivoxil, BMS 200 475 and the other inhibitors mentioned above are still available no generally available clinical experience.

2. the inhibition of HBV by immunological principles, such. B. the treat chronic hepatitis due to interferon; however, it is only moderately effective and has unwanted side effects. Combinations of interferon with lamivudine are not synergistically effective.

Also, the stimulation of our own immune system, such. With thymosin-α already been proposed.

The inhibition by other active substances whose modes of action are unknown or are the subject of speculation, such. B. AT-61 {= N - [(1E) -2-Chloro-2 phenyl-1- (1-piperidinylcarbonyl) -ethenyl] -benzamide}, apparently in the process the packaging of the pregenomic RNA interferes with the unfinished core particles; see. King et al., Antimicrob. Agents and Chemother. 42, 3179-3186 (1998). If Even though the mechanism has not yet been fully elucidated, the authors seem one To exclude effect on the core protein. The in vitro combination of AT-61 and lamivudine is described as being synergistically effective.

Previous therapeutics for the treatment of HBV-infected patients, such. Inter feron or lamivudine, are used as monotherapy. From clinical studies It is known that combinations of both inhibitors have no advantage in the Combat HBV diseases.  

New means for better and effective therapy are therefore desirable.

It has now surprisingly been found that combinations of non- nucleoside inhibitors such as dihydropyrimidines and other HBV-anti viral means the disadvantages of the prior art, or only partially respectively.

The invention therefore relates to combinations A) at least one dihydro pyrimidine and B) at least one of A different HBV antiviral agent, preferably an HBV polymerase inhibitor. The invention therefore relates to combi Nucleosidic and non-nucleoside inhibitors for treatment treatment and prophylaxis of HBV infections and their use for treatment HBV-induced diseases.

The combinations according to the invention inhibit the proliferation of the HBV virus unpredictably much better than those known from the prior art Means or their known combinations. The use of the invention Combinations are valuable in the treatment of HBV-induced diseases Advantages compared to monotherapy with the individual compounds, namely main a synergistic antiviral efficacy, but also a good ver tolerability of the combinations according to the invention in the area of toxicity, in which 50% of the cells survive ("Tox-50") - Compared to the Tox-50 of the single comp components.

Preferred dihydropyrimidines A correspond for example to the formula

or their mesomeric form

wherein
R ^{1 is} phenyl, furyl, thienyl, triazolyl, pyridyl, cycloalkyl having 3 to 6 carbon atoms or radicals of the formulas

where the ring systems listed above are optionally mono- or polysubstituted by identical or different substituents selected from the group consisting of halogen, trifluoromethyl, nitro, cyano, trifluoromethoxy, carboxyl, hydroxyl, (C ₁ -C ₆ ) -alkoxy, (C ₁ - C ₆₎ alkoxycarbonyl and (C ₁ -C ₆₎ - alkyl, substituted, where the alkyl radical can in turn be substituted by aryl having 6 to 10 carbon atoms or halogen,
and the ring systems listed optionally by
-SR ⁶ , -NR ⁷ R ⁸ , -CO-NR ⁹ R ¹⁰ , -SO ₂ -CF ₃ and -A-CH ₂ -R ^{11 are} substituted,
wherein
R ^{6 is} optionally halogen-substituted phenyl,
R ⁷ to R ^{10 are} independently hydrogen, phenyl, hydroxy-substituted phenyl, hydroxy, (C ₁ -C ₆ ) -acyl or (C ₁ -C ₆ ) -alkyl, where the alkyl radical is in turn denoted by hydroxy, (C ₁ -C ₆ ) -Alkoxy carbonyl, phenyl or hydroxy-substituted phenyl,
A is a radical -O-, -S-, -SO- or -SO ₂ -,
R ^{11 is} phenyl which is optionally mono- to polysubstituted, identically or differently, by substituents selected from the group consisting of halogen, nitro, trifluoromethyl, (C ₁ -C ₆ ) -alkyl and (C ₁ -C ₆ ) -alkoxy .
mean,
R ^{2 is} a radical of the formulas -XR ¹² or -NR ¹³ R ¹⁴ ,
wherein
X is a single bond or oxygen,
R ^{12 is} hydrogen, straight-chain or branched (C ₁ -C ₆ ) -alkoxycarbonyl, a straight-chain, branched or cyclic, saturated or unsaturated (C ₁ -C ₈ ) -hydrocarbon radical which optionally has one or two identical or different heterokain members from the group O, -CO-, -NH-, -N- (C ₁ -C ₄ alkyl) -, -S- or -SO ₂ - and optionally substituted by halogen, nitro, cyano, hydroxy, aryl with 6 bis 10 carbon atoms, aralkyl having 6 to 10 carbon atoms, heteroaryl or a group of the formula
-NR ¹⁵ R ^{16 is} substituted,
in which R ¹⁵ and R ¹⁶ independently of one another are hydrogen, benzyl or (C ₁ -C ₆ ) -alkyl,
R ¹³ and R ¹⁴ independently of one another are hydrogen, (C ₁ -C ₆ ) -alkyl or cycloalkyl having 3 to 6 carbon atoms,
R ^{3 is} hydrogen, amino or a radical of the formula

or
Formyl, cyano, hydroxy substituted (C ₁ -C ₆ ) alkylthio, trifluoromethyl or pyridyl or
a straight-chain, branched or cyclic, appropriate, one saturated or unsaturated saturated hydrocarbon radical having up to 8 carbon atoms, which gegebe or more times, identically or differently, by aryloxy having 6 to 10 carbon atoms, azido, halogen, cyano, hydroxy, carboxyl, (C ₁ -C ₆ ) - alkoxycarbonyl, a 5- to 7-membered heterocyclic ring, (C ₁ -C ₆ ) - alkylthio or (C ₁ -C ₆ ) -alkoxy (where the alkylthio or alkoxy radical on its part by azido, amino , Hydroxyl may be substituted) and / or by the group - (CO) _a -NR ¹⁷ R ^{18 is} substituted,
where a is zero or 1,
R ¹⁷ and R ¹⁸ independently of one another are hydrogen or aryl, aralkyl having 6 to 10 carbon atoms or (C ₁ -C ₆ ) -alkyl, where appropriate by (C ₁ -C ₆ ) -alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl where phenyl and benzyl are optionally substituted one or more times, identically or differently, by hydroxyl, carboxyl, (C ₁ -C ₆ ) -alkyl or (C ₁ -C ₆ ) -alkoxy and / or (C ₁ -) C ₆ ) -alkyl is optionally substituted by -NH-CO-CH ₃ or -NH-CO-CF ₃ ,
or
R ¹⁷ and R ^18, together with the nitrogen atom to which they stand, signify a morpholinyl, piperidinyl or pyrrolidinyl ring,
or
R ^{3 is} optionally methoxy-substituted phenyl
or
R ² and R ³ together have a radical of the formula

R ^{4 is} hydrogen, (C ₁ -C ₄ ) -alkyl, (C ₂ -C ₄ ) -alkenyl, benzoyl or acyl having 2 to 6 carbon atoms, preferably hydrogen, methyl, benzoyl or (C ₂ -C ₆ ) -acyl, and
R ^{5 is} pyridyl, pyrimidyl or pyrazinyl, each up to 3-fold, identically or differently, by halogen, hydroxy, cyano, trifluoromethyl, (C ₁ -C ₆ ) -alkoxy, (C ₁ -C ₆ ) -alkyl, (C ₁ -C ₆ ) -alkylthio, carbalkoxy, (C ₁ -C ₆ ) -acyloxy, amino, nitro, mono- or di- (C ₁ -C ₆ ) -alkylamino,
mean,
and their salts.

The compounds I and Ia include the isomers of the formulas (I ') and (Ia) and mixtures thereof. When R ^{4 is} hydrogen, the isomers (I) and (Ia) are in tautomeric equilibrium:

Preference is given to compounds of the formulas (I) or (Ia) in which
R ^{1 is} phenyl, furyl, thienyl, triazolyl, pyridyl, cyclopentyl, cyclohexyl or radicals of the formulas

where the above ring systems are optionally mono- or disubstituted, identical or different, by substituents selected from the group consisting of halogen, trifluoromethyl, nitro, -SO ₂ -CF ₃ , methyl, cyano, trifluoromethoxy, hydroxy, carboxyl, methoxycarbonyl or radicals the formulas
-CO-NH-CH ₂ -C (CH ₃ ) ₃ , -CO-NH (CH ₂ ) ₂ OH,
-CO-NH-CH ₂ -C ₆ H ₅ , -CO-NH-C ₆ H ₅ , -CO-NH- (pOH) -C ₆ H ₄ ,
-O-CH ₂ -C ₆ H ₅ or -S-pCl-C ₆ H _{4 are} substituted,
R ^{2 is} a radical of the formulas -XR ¹² or -NR ¹³ R ¹⁴
wherein
X is a single bond or an oxygen atom,
R is hydrogen, (C ₂ -C ₄₎ alkenyl, (C ₁ -C ₄₎ -alkoxycarbonyl or (C ₁ -C ₄₎ - alkyl, where the alkyl radical, where appropriate, by pyridyl, cyano, phenoxy, benzyl or by a radical Formula -NR ¹⁵ R ^{16 is} substituted,
wherein
R ¹⁵ and R ¹⁶ independently of one another denote hydrogen, benzyl or (C ₁ -C ₄ ) -alkyl,
R ¹³ and R ¹⁴ independently of one another are hydrogen, (C ₁ -C ₄ ) -alkyl or cyclopropyl,
R ^{3 is} hydrogen, amino or a radical of the formula

or
Formyl, cyano, hydroxy-substituted (C ₁ -C ₄ ) -alkylthio, trifluoromethyl, cyclopropyl, pyridyl or (C ₁ -C ₄ ) -alkyl, where the alkyl radical is optionally substituted by halogen, (C ₁ -C ₄ ) -alkoxycarbonyl, Hydroxy and / or through the group
- (CO) _a -NR ¹⁷ R ^{18 is} substituted,
wherein
a zero or 1,
R ¹⁷ and R ¹⁸ independently of one another are hydrogen, phenyl, benzyl or (C ₁ -C ₄ ) -alkyl, which are optionally substituted by (C ₁ -C ₃ ) -alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl, where independently one another, phenyl and benzyl optionally mono- or disubstituted by identical or different manner by hydroxyl, carboxyl, (C ₁ -C ₃₎ - alkyl or (C ₁ -C ₃₎ -alkoxy and (C ₁ -C ₃₎ alkyl ge optionally substituted by radicals of the formulas -NH-CO-CH ₃ or -NH-CO-CF ₃ , phenyl or
R ¹⁷ and R ^18, together with the nitrogen atom to which they stand, signify a morpholinyl, piperidinyl or pyrrolidinyl ring,
or
R ^{3 is} optionally methoxy-substituted phenyl
or
R ² and R ³ together have a radical of the formula

R ^{4 is} hydrogen, methyl, vinyl or acetyl and
R ^{5 is} pyridyl, pyrimidyl or pyrazinyl,
and their salts.

Particular preference is given to compounds of the formulas (I) and (Ia)
wherein
R ^{1 is} phenyl, furyl, thienyl, triazolyl, pyridyl, cyclopentyl, cyclohexyl or radicals of the formulas

wherein the ring systems listed above are optionally up to twice, identically or differently, by substituents selected from among fluorine, chlorine, bromine, iodine, hydroxyl, trifluoromethyl, nitro, -SO ₂ -CF ₃ , methyl, cyano, trifluoromethoxy, carboxyl , Methoxycarbonyl or radicals of the formulas -CO-NH-CH ₂ -C (CH ₃ ) ₃ , -CO-NH (CH ₂ ) ₂ OH, -CO-NH-CH ₂ -C ₆ H ₅ , -CO-NH- C ₆ H ₅ , -CO-NH- (pOH) -C ₆ H ₄ , -O-CHrC ₆ H ₅ or -S-pCl-C ₆ H _{4 are} substituted,
R ^{2 is} a radical of the formulas -XR ¹² or -NR ¹³ R ¹⁴ ,
wherein
X is a single bond or an oxygen atom,
R ¹² is hydrogen, (C ₂ -C ₃₎ alkenyl, (C ₁ -C ₄₎ -alkoxycarbonyl or (C ₁ -C ₄₎ - alkyl, wherein the alkyl moiety is optionally substituted by pyridyl, cyano, phenoxy, benzyl or by a radical the formula -NR ¹⁵ R ^{16 is} substituted,
wherein
R ¹⁵ and R ¹⁶ independently of one another denote hydrogen or methyl,
R ¹³ and R ¹⁴ independently of one another are hydrogen, (C ₁ -C ₃ ) -alkyl or cyclopropyl,
R ^{3 is} hydrogen, amino or a radical of the formula

or
Formyl, cyano, trifluoromethyl, cyclopropyl, pyridyl or (C ₁ -C ₄ ) -alkyl be, where the alkyl radical is optionally substituted by fluorine, chlorine, (C ₁ -C ₃ ) - alkoxycarbonyl or hydroxy,
or
R ^{3 is} optionally methoxy-substituted phenyl or
R ² and R ³ together have a radical of the formula

R ^{4 is} hydrogen, methyl, vinyl or acetyl and
R ^{5 is} pyridyl, pyrimidyl or pyrazinyl
mean,
and their salts.

Very particular preference is given to compounds of the formulas (I) and (Ia) in which
R ^{1 is} phenyl or triazolyl, which are optionally substituted up to twice, identically or differently by fluorine, chlorine, bromine or iodine,
R ² straight-chain or branched alkoxycarbonyl having up to 4 carbon atoms,
R ^{3 is} methyl, ethyl or cyclopropyl or
R ² and R ³ together have a radical of the formula

R ^{4 is} hydrogen, vinyl or acetyl and
R ^{5 is} pyridyl
mean.

Specifically preferred dihydropyrimidines A are listed in Table A.  

Table A

Very particular preference is given to the dihydropyrimidines A listed in Table B.

Table B

Very particular preference is given to the following compounds:

their mesomeric forms and their salts.

The compounds of the formulas (I), (Ia) or Tables A and B can be prepared by

[A] Aldehydes of the formula

R ¹ -CHO (II)

wherein R ^{1 has} the meaning given above,
with amidines of the formula

wherein R ^{5 has} the meaning given above,
and compounds of the formula

R ³ -CO-CH ₂ -CO-R ² (IV)

wherein R ² and R ^{3 have} the meanings given above,
if appropriate in the presence of inert organic solvents with or without base or acid addition,
or
[B] Compounds of the formula

wherein R ¹ to R ^{3 have} the meanings given above,
with amidines of the formula

wherein R ^{5 has} the meaning given above,
if appropriate in the presence of inert organic solvents at temperatures of 20 ° C. to 150 ° C. with or without addition of base or acid,
or
[C] aldehydes of the formula

R ¹ -CHO (II)

wherein R ^{1 has} the meaning given above,
with compounds of the formula

wherein R ² and R ^{3 have} the meanings given above,
and amidines of the formula (III) as described above,
or
[D] Aldehydes of the formula (II) with compounds of the formula (IV) and iminoethers of the formula

in which R ^{5 has} the abovementioned meaning and R ^{1 is} (C ₁ -C ₄ ) -alkyl,
in the presence of ammonium salts.

The preferred method [A] can be exemplified by the following formula scheme:
[A]

For all process variants A, B, C and D, all inert solvents are used organic solvents in question. These include, preferably, alcohols such as Ethanol, methanol, isopropanol, ethers, such as diethyl ether, tetrahydrofuran, dioxane,  Glycol monomethyl ether, glycol dimethyl ether, carboxylic acids such as glacial acetic acid, or Dimethylformamide, dimethyl sulfoxide, acetonitrile, pyridine and hexamethyl triamide.

The reaction temperatures can be varied within a wide range become. In general, one works in the range of 20 to 150 ° C, preferably but at the boiling point of the respective solvent.

The reaction can be carried out at normal pressure, but also at elevated pressure become; in general, one works under normal pressure.

The reaction can be carried out with or without base or acid addition; the presence of weaker acids, such as. Acetic or formic acid, is preferred.

The aldehydes (II) used as starting materials are known or can after be prepared by literature methods [cf. T.D. Harris and G.P. Roth, J. Org. Chem. 44, 146 (1979), DE-OS 21 65 260 and 24 01 665, Mijano et al., Chem. Abstr. 59, (1963), 13,929c, E. Adler and H.-D. Becker, Chem. Scand. 15, 849 (1961), E.P. Papadopoulos, M. Mardin and Ch. Issidoridis, J. Org. Chem. Soc. 78 2543 (1956)).

The β-ketocarboxylic acid esters (IV) used as starting materials are known or can be prepared by literature methods [z. B. Borrmann, "Implementation of diketene with alcohols, phenols and mercaptans" in "Methods of Organic Chemistry "(Houben-Weyl), Vol. VII / 4, 230 ff (1968); Y. Oikawa, K. Sugano and O. Yonemitsu, J. Org. Chem. 43, 2087 (1978)].

The ylidene-β-keto esters (V) used as starting materials can according to literature be prepared known methods [see. G. Jones, "The Knoevenagel Conden sation "in Organic Reactions, Vol. XV, 204 ff. (1967)].  

The enaminocarboxylic esters (VI) used as starting materials and the imino ethers (VII) are known or can be prepared by literature methods be [cf. S.A. Glickman and A.C. Cope, J. Am. Chem. Soc. 67, 1017 (1945)].

The compounds (III) can be prepared by reacting compounds of the formula

R ⁵ -CN (VIII)

wherein R ^{5 has} the meaning given above, as usual on the imino ethers and finally with ammonium chloride in methanol [cf. see WK Fife, Heterocycles 22, 93-96 (1984); T. Sakamoto, S. Kaneda, S. Nishimura, H. Yamanaka, Chem. Pharm. Bull. 33, 565, 571 (1986)] or other methods known in the literature, such as. Garigipati, Tetrahedron Lett. 1990, 1969-1972, Boere et al., J. Organomet. Chem. 1987, 331, 161, or Caton et al., J. Chem. Soc. 1967, 1204.

All process steps can be carried out at normal pressure and in a temperature range from 0 to 130 ° C, preferably from 20 to 100 ° C, take place.

The compounds (VIII) are known or can be prepared by processes known per se, for example pyridines of the formula

R ⁵ -H (IX)

wherein the hydrogen is ortho to nitrogen and R ^{5 is} as defined above,
initially at 50 to 150 ° C, preferably at about 100 ° C, in H ₂ O ₂ / glacial acetic converts to the corresponding N-oxides and then with trimethylsilyl cyanide (TMSCN) by literature methods, for example in the above listed inert solvents, preferably Acetonitrile, THF, toluene at room temperature or at reflux temperature, optionally with the addition of bases such as triethylamine or DBU, is reacted
or by being in compounds of the formula

in which Y and Z represent the substitution radicals of the pyridyl ring indicated under R,
with the help of cyanides, such as potassium cyanide or copper cyanide, which exchanges chlorine for cyanide,
or in the case where R ^{5 is} difluoropyridyl, compounds of the formula

wherein Y 'and Z' independently of one another are chlorine or bromine,
with alkali metal or ammonium fluorides, preferably potassium fluoride, literature methods known in the literature in polar solvents, such as polyglycols, poly glycol ethers, DMSO or sulfolane, optionally with the addition of phase transfer catalysts, in the sense of a halogen / fluorine exchange reaction.

The above process is exemplified with respect to the 3,5-difluoropyridyl compounds by the following reaction scheme:

The above compounds I and Ia and various processes for their preparation are from DE-OS 198 17 264 (= WO 99/54 326) and 198 17 265th (= WO 99/54 312) known.

Further preferred dihydropyrimidines A correspond to the formula

or their isomeric form

wherein
R ^{1 is} phenyl, furyl, thienyl, pyridyl, cycloalkyl of 3 to 6 carbon atoms or a radical of the formulas

where the ring systems listed above are optionally mono- or polysubstituted by identical or different substituents selected from the group consisting of halogen, trifluoromethyl, nitro, cyano, trifluoromethoxy, carboxyl, hydroxyl, (C ₁ -C ₆ ) -alkoxy, (C ₁ - C ₆₎ alkoxycarbonyl and (C ₁ -C ₆₎ - alkyl, substituted, where the alkyl radical can in turn be substituted by aryl having 6 to 10 carbon atoms or halogen, and / or the ring systems mentioned are optionally substituted by groups of the formulas -SR ⁶ , -NR ⁷ R ⁸ , -CO-NR ⁹ R ¹⁰ , SO ₂ -CF ₃ and -A-CH ₂ -R ^{11 are} substituted,
wherein
R ^{6 is} optionally halogen-substituted phenyl,
R ⁷ to R ¹⁰ independently of one another are hydrogen, phenyl, hydroxy-substituted phenyl, hydroxyl, (C ₁ -C ₆ ) -acyl or (C ₁ -C ₆ ) -alkyl, where the alkyl radical is in turn replaced by hydroxyl, (C ₁ - C ₆ ) -alkoxycarbonyl, phenyl or hydroxy-substituted phenyl may be substituted,
A is a radical -O-, -S-, -SO- or -SO ₂ -,
R ^{11 is} phenyl which is optionally mono- to polysubstituted by identical or different substituents selected from the group consisting of halogen, nitro, trifluoromethyl, (C ₁ -C ₆ ) -alkyl and (C ₁ -C ₆ ) -alkoxy, mean,
R ^{2 is} a radical of the formulas -OR ¹² or -NR ¹³ R ¹⁴ ,
wherein
R ^{12 is} hydrogen, (C ₁ -C ₆ ) -alkoxycarbonyl or a straight-chain, branched or cyclic, saturated or unsaturated (C ₁ -C ₈ ) hydrocarbon radical which optionally has one or two identical or different heterokain members from the group -O- , CO-, -NH-, -N- (C ₁ -C ₄ alkyl) -, -S- and -SO ₂ - and optionally substituted by halogen, nitro, cyano, hydroxy, aryl having 6 to 10 carbon atoms or Aralkyl having 6 to 10 carbon atoms, heteroaryl or a group of the formula -NR ¹⁵ R ^{16 is} substituted,
in which R ¹⁵ and R ¹⁶ independently of one another are hydrogen, benzyl or (C ₁ -C ₆ ) -alkyl,
R ¹³ and R ¹⁴ independently of one another are hydrogen, (C ₁ -C ₆ ) -alkyl or cycloalkyl having 3 to 6 carbon atoms,
R ^{3 is} hydrogen, amino or a radical of the formula

or formyl, cyano, hydroxy-substituted (C ₁ -C ₄ ) -alkylthio, trifluoromethyl or
a straight-chain, branched or cyclic, saturated or unge saturated hydrocarbon radical having up to 8 carbon atoms, where appropriate, one or more times, identically or differently by aryloxy having 6 to 10 carbon atoms, azido, cyano, hydroxyl, carboxyl, (C ₁ -C ₆ ) -alkoxycarbonyl, a 5- to 7-membered heterocyclic ring, (C ₁ -C ₆ ) -alkylthio or (C ₁ -C ₆ ) -alkoxy (where the alkylthio or alkoxy radical is in turn linked by azido, amino or hydroxyl may be substituted) and / or is substituted by the group - (CO) _a -NR ¹⁷ R ¹⁸ ,
where a is zero or 1,
R ¹⁷ and R ¹⁸ independently of one another are hydrogen or aryl, aralkyl having 6 to 10 carbon atoms or (C ₁ -C ₆ ) -alkyl, where appropriate by (C ₁ -C ₆ ) -alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl where phenyl and benzyl are optionally substituted one or more times, identically or differently, by hydroxyl, carboxyl, (C ₁ -C ₆ ) -alkyl or (C ₁ -C ₆ ) -alkoxy and / or (C ₁ -) C ₆ ) -alkyl is optionally substituted by -NH-CO-CH ₃ or NH-CO-CF ₃ ,
or
R ¹⁷ and R ^18, together with the nitrogen atom to which they stand, signify a morpholinyl, piperidinyl or pyrrolidinyl ring,
D is an oxygen or sulfur atom and
R ^{5 is} hydrogen, halogen or straight-chain or branched alkyl having up to 6 carbon atoms
mean,
and their salts.

Preference is given to compounds of the formulas I or Ia,
wherein
R ^{1 is} phenyl, furyl, thienyl, pyridyl, cyclopentyl or cyclohexyl, these ring systems optionally one or two times, identically or differently, by substituents selected from the group halogen, trifluoromethyl, nitro, SO ₂ -CF ₃ , methyl, cyano, trifluoromethoxy , Carboxyl, methoxycarbonyl or radicals of the formulas -CO-NH-CH ₂ -C (CH ₃ ) ₃ , -CO-NH (CH ₂ ) ₂ OH, CO-NH-CH ₂ -C ₆ H ₅ , -CO-NH -C ₆ H ₅ , -CO-NH- (pOH) -C ₆ H ₄ , -O-CH ₂ -C ₆ H ₅ or -S-pCl-C ₆ H _{4 are} substituted,
R ^{2 is} a radical of the formulas -OR 2 or -NR ¹³ R ¹⁴ ,
wherein
R ^{12 is} hydrogen, (C ₂ -C ₄ ) -alkenyl or (C ₁ -C ₄ ) -alkyl, where the alkyl radical is in turn optionally substituted by pyridyl, cyano, phenoxy, benzyl or by a radical of the formula -NR ¹⁵ R ¹⁶ in which R ¹⁵ and R ^16, independently of one another, denote hydrogen, methyl or ethyl,
R ¹³ and R ¹⁴ independently of one another are hydrogen, methyl, ethyl or cyclopropyl,
R ^{3 is} hydrogen or a radical of the formula

or
Formyl, cyano, trifluoromethyl, cyclopropyl or (C ₁ -C ₄ ) -alkyl, where the alkyl radical is in turn optionally substituted by radicals of the formulas SO ₂ CH ₃ , -NH-CO-CH ₃ , NH-CO-CF ₃ , fluorine, Chlorine, (C ₁ -C ₃ ) -alkoxycarbonyl, hydroxy and / or is substituted by the group - (CO) a -NR ¹⁷ R ¹⁸ ,
wherein
a zero or 1,
R ¹⁷ and R ^{18 are} independently hydrogen, phenyl, benzyl or (C ₁ -C ₄ ) -alkyl optionally substituted by (C ₁ -C ₃ ) -alkoxy carbonyl, amino, hydroxyl, phenyl or benzyl, wherein independently one another, phenyl and benzyl optionally mono- or disubstituted by identical or different manner by hydroxyl, carboxyl, (C ₁ -C ₃₎ -alkyl or (C ₁ -C ₃₎ -alkoxy and (C ₁ -C ₃₎ - alkyl optionally is substituted by radicals of the formulas -NH-CO-CH ₃ or NH-CO-CF ₃ , phenyl or
R ¹⁷ and R ^18, together with the nitrogen atom to which they stand, signify a morpholinyl, piperidinyl or pyrrolidinyl ring,
R ^{4 is} hydrogen, methyl, ethyl or acetyl,
D is an oxygen or sulfur atom and
R ^{5 is} hydrogen, halogen or (C ₁ -C ₄ ) -alkyl
mean,
and their salts.

Particular preference is given to compounds of the formulas I and Ia,
wherein
R ^{1 is} phenyl or thienyl, these ring systems being optionally substituted up to twice, identically or differently, by substituents selected from among fluorine, chlorine, methyl, trifluoromethyl and nitro,
R ^{2 is} a radical of the formulas -OR ¹² or -NR ¹³ R ¹⁴ ,
wherein
R ^{12 is} hydrogen or (C ₁ -C ₄ ) -alkyl and
R ¹³ and R ¹⁴ independently of one another denote hydrogen or methyl,
R ^{3 is} hydrogen or formyl, cyano, trifluoromethyl, cyclopropyl or where appropriate by fluorine, chlorine, hydroxy substituted (C ₁ -C ₃ ) alkyl, which in turn may be substituted up to 3-fold by C ₁ -C ₃ alkoxycarbonyl .
R ^{4 is} hydrogen or methyl,
D is an oxygen or sulfur atom and
R ^{5 is} hydrogen, fluorine, chlorine or (C ₁ -C ₃ ) -alkyl
mean,
and their salts.

Very particular preference is given to compounds of the formulas I and Ia in which
R ^{1 is} phenyl or thienyl, which are optionally substituted up to twice, identically or differently by fluorine or chlorine,
R ^{2 is} methoxy, ethoxy or n-propoxy,
R ^{3 is} hydrogen, methyl or cyclopropyl,
R ^{4 is} hydrogen,
D is an oxygen or sulfur atom and
R ^{5 is} hydrogen, fluorine or chlorine
mean,
and their salts.

Cycloalkyl having 3 to 6 carbon atoms in the context of the invention is cyclo propyl, cyclopentyl, cyclobutyl, cyclohexyl, preferably cyclopentyl and cyclo hexyl.

Aryl is generally an aromatic radical having 6 to 10 carbon atoms, preferably phenyl and naphthyl.

Aralkyl is in the context of the invention for aralkyl preferably having 6 to 10, in particular 6 carbon atoms in the aryl part (preferably phenyl or naphthyl, in particular phenyl) and preferably 1 to 4, in particular 1 or 2 carbon atoms in the alkyl moiety, wherein the alkyl moiety may be linear or branched. preferred Aralkyl radicals are benzyl and phenethyl.

Heteroaryl is in the context of the invention for 5- to 7-membered rings with preference Example 1 to 3, in particular 1 or 2 identical or different heteroatoms of the Series oxygen, sulfur and nitrogen. Preferred examples include furyl, Thiophenyl, pyrazolyl, imidazolyl, 1.2.3- and 1.2.4-triazolyl, oxazolyl, isoxazolyl, Thiazolyl, isothiazolyl, 1.2.3-, 1.3.4-, 1.2.4- and 1.2.5-oxadiazolyl, pyrrolyl, pyridyl, Pyrimidinyl, pyrazinyl, 1.3.5-, 1.2.4- and 1.2.3-triazinyl, 1.2.4-, 1.3.2-, 1.3.6- and 1.2.6-oxazinyl.

Acyl in the context of the invention is a straight-chain or branched acyl radical with 1 to 6, preferably 1 to 4 carbon atoms, such as. Acetyl and propionyl.

Alkyl in the context of the invention is a straight-chain or branched alkyl rest with 1 to 6, preferably 1 to 4 carbon atoms, such as. Methyl, ethyl, Propyl, isopropyl, tert -butyl, n -pentyl and n -hexyl.  

Alkenyl is in the context of the invention for a straight-chain or branched Alkenyl radical having 2 to 6, preferably 3 to 5 carbon atoms, such as. Ethenyl, Propenyl, isopropenyl, tert.Butenyl, n-pentenyl and n-hexenyl.

Alkoxy in the context of the invention is a straight-chain or branched one Alkoxy radical having 1 to 6, preferably 1 to 4 carbon atoms, such as. Methoxy, Ethoxy, propoxy, isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy.

Alkylthio in the context of the invention is a straight-chain or branched one Alkylthio radical having 1 to 6, preferably 1 to 4, carbon atoms, such as Methylthio, ethylthio and propylthio.

Alkoxycarbonyl is in the context of the invention for a straight-chain or branched alkoxycarbonyl radical having 1 to 6, preferably 1 to 4 carbon atoms, such as Methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, tert-butoxycarbonyl, n-pentoxycarbonyl and n-hexoxycarbonyl.

Halogen is in the context of the invention for fluorine, chlorine, bromine or iodine.

The compounds I and Ia can in stereoisomeric forms, either as Image and mirror image (enantiomers) or not like image and mirror image (Diastereomers) behave exist. The compounds I and Ia thus include so probably the enantiomers as well as the diastereomers and their respective Mixtures. The racemic forms can be as well as the diastereomers in be can be separated into the stereoisomerically uniform components.

The compounds I and Ia can also be present as salts. As part of the Er Physically acceptable salts are preferred.

Physiologically acceptable salts may be salts of the compounds I or Ia with inorganic or organic acids. Preference is given to salts of inorganic  Acids, such as hydrochloric, hydrobromic, phosphoric or Sulfuric acid, or salts of organic carboxylic or sulfonic acids, such as acetic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, Lactic acid, benzoic acid, or methanesulfonic acid, ethanesulfonic acid, phenylsulfone acid, toluenesulfonic acid or naphthalenedisulfonic acid.

Physiologically acceptable salts may also be metal or ammonium salts of Compounds I or Ia be. Particular preference is z. For example, sodium, potassium, Magnesium or calcium salts and ammonium salts of ammonia or organic amines, such as ethylamine, di- or triethylamine, di- or Triethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine, lysine, ethylene diamine or 2-phenylethylamine.

The compounds of the formula I or Ia can be prepared by
[A] Aldehydes of the formula

R ¹ -CHO (II)

wherein R ^{1 has} the meaning given above, with amidines or their hydrochlorides of the formula

wherein R ⁵ and D have the meanings given above,
and compounds of the formula

R ³ -CO-CH ₂ -CO-R ² (IV)

in which R ² and R ^{3 have} the abovementioned meanings, if appropriate in the presence of inert organic solvents with or without base or acid addition,
or
[B] Compounds of the formula

wherein R ¹ to R ^{3 have} the meanings given above,
with amidines of the formula

wherein R ⁵ and D have the meanings given above,
if appropriate in the presence of inert organic solvents at temperatures between 20 ° C. and 150 ° C. with or without addition of base or acid,
or
[C] aldehydes of the formula

R ¹ -CHO (II)

wherein R ^{1 has} the meaning given above, with compounds of the formula

wherein R ² and R ^{3 have} the meanings given above,
and amidines of the formula (III) as described above.

The preparation processes can be exemplified by the following equation schemes:

For all process variants A, B and C come as solvent all inert orga niche solvents in question. These include, preferably, alcohols, such as ethanol,  Methanol, isopropanol, ethers, such as dioxane, diethyl ether, tetrahydrofuran, glycol monomethyl ether, glycol dimethyl ether, carboxylic acids such as glacial acetic acid, or dimethyl formamide, dimethyl sulfoxide, acetonitrile, pyridine and hexamethylphosphoric tri amide.

The reaction temperatures can be varied within a wide range become; in general, one works between 20 and 150 ° C, but preferably at the boiling point of the particular solvent.

The reaction can be carried out at atmospheric pressure, but also at elevated pressure become; in general, one works under normal pressure.

The reaction can be carried out with or without base or acid addition, preferably in Presence of weaker acids, such as. As acetic acid or formic acid, by be guided.

The aldehydes (1I) used as starting materials are known or can after be prepared by literature methods [vgl. T.D. Harns and G.P. Roth, J. Org. Chem. 44, 146 (1979), DE-OS 21 65 260 and 24 01 665, Mijano et al., Chem. Abstr. 59, (1963), 13,929c, E. Adler and H.-D. Becker, Chem. Scand. 15, 849 (1961), E.P. Papadopoulos, M. Mardin and Ch. Issidoridis, J. Org. Chem. Soc. 78 2543 (1956)].

The amidines (III) used as starting materials are known or can after be prepared by literature methods [cf. "Methods of the organic Chemistry "(Houben-Weyl), Vol. 11/2, page 38 ff (1958), R.L. Shoiner and F.W. Neumann, Chem. Review 35, 351 (1944)].

The β-ketocarboxylic acid esters (IV) used as starting materials are known or can be prepared by literature methods [z. B. Borrmann, "Implementation of diketene with alcohols, phenols and mercaptans", in "Methods  of Organic Chemistry "(Houben-Weyl), Vol. VII / 4, 230 ff (1968); Y. Oikawa, K. Sugano and O. Yonemitsu, J. Org. Chem. 43, 2087 (1978)].

The ylidene-β-keto esters (V) used as starting materials can according to literature be prepared known methods [see. G. Jones, "The Knoevenagel Conden sation "in Organic Reactions, Vol. XV, 204 ff. (1967)].

The enaminocarboxylic esters (VI) used as starting materials are known or can be prepared by literature methods [cf. A.C. Cope, J. At the. Chem. Soc. 67, 1017 (1945)].

The compounds I and Ia, which in the 2-position optionally substituted Oxazolyl or thiazolyl, and various methods for their Her position are known from DE-OS 198 17 262 (= WO 99/54 329).

The dihydropyrimidines A act as HBV core protein inhibitors.

Another object of the invention are therefore combinations A) at least an HBV core protein inhibitor and B) at least one of A different HBV antiviral agent, preferably at least one HBV polymerase Inhibitor.

HBV core protein inhibitors according to the invention are those non-nucleosomes dical inhibitors present in the cell (i) the half-life of the HBV core protein at least halve or (ii) the binding assay described below consist.  

(i) Pulse chase labeling of HepG2.2.15 cells and subsequent Detection of the HBV core protein by immunoprecipitation general description

By pulse-chase labeling of HepG2.2.15 cells and subsequent immunoprecipitation of the HBV core protein, the rate of synthesis and the stability of the HBV core protein can be measured. As a measure of stability, the half-life of the core protein is used. The half-life of the core protein is defined by the time within which the core protein amount has been reduced to half the amount present at the initial time t ₀ = 0.

The amount of protein correlates with the incorporated radioactivity and consequently the Blackening of an X-ray film or radiation intensity caused by a phos phorimager can be made visible.

With the help of this technique, the influence of potential inhibitors on the half-life time of the viral HBV core protein. A substance is accordingly then an HBV core protein inhibitor, when the half-life of the HBV core protein at least halved; see. subsequent test description.

Experimental Procedure Seeding of the cells

On day 1, the HepG2.2.15 cells are placed in cell culture flasks (75 cm ² , Costar) at a density of 6 × 10 ⁶ cells per bottle in HepG2 cell culture medium (Biochrom KG, Berlin). 100 liters of this medium consist of 74.8 liters of ®seromed RPMI 1640 medium and 14.4 liters of ®seromed Medium 199 with Earle's salts (both media from Biochrom KG, Berlin, the compositions are described before the examples); Furthermore, 200 g of NaHCO ₃ , 5 000 000 units of penicillin G sodium, 5 g of streptomycin, 25 mg of amphotericin B, 30 g of aminoglycoside antibiotic G418 (Sigma, Munich), aqueous sodium pyruvate solution (up to a sodium pyruvate solution). Concentration of the total solution of 2 mM), aqueous glutamine solution (up to a glutamine concentration of the total solution of 2 mM), aqueous FCS (FCS = fetal calf serum, up to an FCS concentration of the total solution of 2% (v / v )). The test substance is prepared as a 50 mM stock solution in dimethylsulfoxide (DMSO, Sigma, Munich). It is diluted in HepG2 cell culture medium and adjusted to 100-fold IC ₅₀ concentration. 1 ml of this test substance solution is added to 19 ml HepG2 cell culture medium. The test substance is added with the sowing in graduated dilution steps. The cells are incubated at 37 ° C and 5% CO ₂ (v / v) in the incubator.

Labeling of the proteins

On day 3, the culture medium is discarded and replaced with 20 ml of a medium deprived of the amino acids methionine and cystine ("starvation medium"). The starvation medium (500 ml) contains MEM (= minimum essential medium with Earle's salts, Gibco 51091-015 - the composition is described before the examples), 5 ml aqueous L-glutamine solution (200 mM), 5 ml L-aqueous Leucine solution (5.2 g / l), 5 ml aqueous glucose solution (100 g / l), 5 ml aqueous i-inositol solution (0.2 g / l), 5 ml aqueous L-arginine solution (12.46 g / l). l,) and aqueous FCS (up to a total solution FCS concentration of 2% v / v). Incubation with starvation medium is carried out in the presence of the test substance (concentration as above). The cells are incubated for 45 minutes at 37 ° C and 5% CO ₂ (v / v) in the incubator. Subsequently, 16 ml of starvation medium are taken off and the remaining medium is mixed with 25 μl of ³⁵ S Redivue Promix (2.5 mCi / 175 μl) (Amersham, AGQ 0080). The cells are incubated for a further 10 minutes at 37 ° C. and 5% CO ₂ (v / v) in the incubator ("pulses"). The labeling solution is then removed and replaced with starvation medium plus aqueous L-cystine solution (up to a cystine concentration of the total solution of 1 mM) and aqueous L-methionine solution (up to a methionine concentration of the total solution of 1 mM). ("Labelstopmedium") replaced. This stops the marker. The incubation with label stop medium is also carried out in the presence of the test substance (concentration as above). The total volume is 20 ml per bottle. The cultures are incubated at 37 ° C. and 5% CO ₂ (v / v) in the incubator ("chase"). 5 minutes after the label stop medium has been added, the time t ₀ = 0 ("0 hour value") is taken. Further experimental times are variable in the range up to 96 hours. At each additional time value in the course of the "chase", a sample set (treated / untreated) is taken to determine the core protein content at this time. The medium is removed and the cells are washed once with PBS (phosphate-buffered saline without Ca / Mg ⁺⁺ ) (Biochrom No. L1825). Subsequently, the cell lawn is washed with 1 ml lysis buffer {10 mM Tris-HCl pH 7.5; 1% (v / v) of the nonionic detergent ®Triton X-100 [trademark of Rohm & Haas, 4- (1.1.3.3-tetramethylbutyl) phenol-polyethoxyethanol]; Lysed 140 mM NaCl}. (The term "tris" means 2-amino-2- (hydroxymethyl) -1,3-propanediol). The lysate is transferred to 2 ml Eppendorf tubes and centrifuged (2 minutes, 4000 rpm). The supernatant is transferred to a new Eppendorf tube and the sediment (= cell nuclei) is discarded. The lysate is stored at + 4 ° C.

Precipitation of the HBV core protein by specific immunoprecipitation

To the lysate are added 10 μl normal rabbit serum (Sigma 7523) and 200 μl Sepharose beads suspended in PBS (Protein A Sepharose C1-4B; Pharmacia, 17-0780-01) (Alternatively, the complex of beads and antibodies are preformed). This is followed by incubation for 2 hours at 4 ° C on the wheel. The immunoprecipitates are separated by centrifugation (2 minutes, 1000 rpm) and stored at 4 ° C. The supernatant will turn into a new one Transferred Eppendorf tube.  

Subsequently, 5 μl of anti-human HBV core rabbit antiserum and 200 μl Sepharose beads suspended in PBS (Protein A Sepharose C1-4B; Pharmacia, 17-0780-01) (Alternatively, the complex may be Beads and antibodies are preformed). This is followed by incubation (2 hours at 4 ° C) on the wheel. The immunoprecipitates are made by centrifugation separated (2 minutes, 1000 rpm) and stored at 4 ° C. The supernatant is in a transferred new Eppendorf tube and stored at 4 ° C.

The beads are washed three times with NET buffer [50 mM Tris-HCl pH 7.4; 0.5% (v / v) nonionic detergent ® Nonident NP-40 (Boehringer Mannheim); 150 mM NaCl; 5 mM EDTA (ethylenediaminetetraacetic acid)), d. H. from alternately suspended and centrifuged (2 minutes at 1000 rpm). The ge Washed immunoprecipitates are stored at 4 ° C.

SDS-page and visualization of radiolabelled HBV-core proteins (SDS = Dodecyl sulfate sodium)

The washed immunoprecipitates are replaced by 12% by weight SDS-polyacrylamide Gel electrophoresis (SDS-PAGE) separated. For this purpose, each sample with 50 ul twice concentrated SDS sample buffer (BioRad), once vortexed (shaken), boiled for 5 minutes at 95 ° C, quenched on ice and briefly centri fugue. In each gel pocket 20 ul of the supernatant are applied. The gel is running 1.5 hours at 120 volts (= 15 volts / cm).

The gel is placed three times for 5 minutes in fixing solution (70 vol.% Ethanol, 10 vol. Acetic acid; 20% by volume of water). Subsequently, the gel for 20 minutes in enhancer (Enlightning Rapid Autoradiography Enhancer NEN Research Products No. NEF-974). The gel is placed on filter paper (BioRad No. 165-0921) and covered with cellophane (BioRad No. 165-0922). These Arrangement is dried for 50 minutes with the BioRad gel dryer.  

The dried gel is applied to an X-ray film (Hyperfilm MP, Amersham RPN 2115H) and developed after a reasonable period of exposure. Alternatively, you can The dried gel can be analyzed on a phosphorimager (Fujix BAS 100). The evaluation compares the half-lives of the HBV core protein in on and off Absence of the test substance.

(ii) Gel chromatographic detection of the binding of a labeled test substance to HBV core protein general description

The chromatographic behavior of a protein or one of its natural occurring aggregation forms (dimers, multimers) during gel permeation Chromatography depends on the choice of gel material used. example For example, the gel material used in desalination columns (e.g. Sephadex G-25; Pharmacia), have an exclusion limit for globular proteins is at a molecular weight of 5000 daltons; d. H. all proteins with one Molecular weight greater than 5000 Da elutes in the exclusion volume.

On the other hand, a globular protein that has a molecular weight below 5000 Da penetrates in the release material and is retained on the column. One sees effects not determined by molecular size (eg adsorption), As a rule, the lower the molecular weight of a molecule, the better the path through the gel bed lasts longer and the later the molecule is eluted. This basic experimental arrangement can be used for a binding test. The binding test is used to examine whether a test substance binds to a protein of Interest binds. For this, the test substance, which is sufficiently below the exclusion must be marked (eg by replacing a hydrogen atom with Tritium) and with the protein (e.g., the HBV core protein or its aggregates form = "capsid"), which must be sufficiently above the exclusion limit, incubated. The material is applied to the column and washed with an elution buffer (eg. 10 mM Tris pH 7.4 and 1 mM EDTA). By binding to the protein  the labeled test substance is eluted as the free protein in the exclusion volume. A non-binding test substance or the test substance excess, however, is in eluted eluting late. To prove the elution behavior individual fractions collected and the radioactivity in the scintillation counter (or determined by another suitable detection method).

In general, this method is carried out as a variant of the binding test be that, for example, a labeled substance that binds to the HBV core Protein binds ("labeled substance"), used to make more core protein binders To find: The labeled substance is replaced by a test substance ("displacing Substance ") directly displaced (competition for the same binding site) or the Test substance modifies the protein (or one of its aggregate forms) in such a way that the labeled substance can no longer bind (modification of the binding site).

Experimental Procedure Provision of the column

The Sephadex G-25 column (column height: 5 cm, column diameter: 1.5 cm) is cast according to standard procedures (manufacturer's instructions) or finished by to a manufacturer (e.g., PD10 desalting columns from Phamacia). The pillar is equilibrated in the desired buffer (10 mM Tris pH 7.4 and 1 mM EDTA). One sample (32 μl) consisting of 2 μl (= 64 μmol) of labeled substance solution (specific activity of a tritiated compound, for example, is typically mCi / ml range) mixed with HBV-core protein (10 μg = 2.7 μmol), which in the form of the capsid, and with displacing substance (100 × molar excess, based on labeled substance) is applied to the column. The equilibration buffer is used for elution. Fractions of 1 ml are collected. The entire fraction or an aliquot thereof is washed with scintillator fluid (Ultima Gold, Packard) and measured with a scintillation counter. In the case a displacement of labeled substance by the test substance is in the Fractions containing the exclusion volume have a lower activity than in  Measured fractions in which no test substance was present (zero value). An HBV core Protein inhibitor is present when a maximum of 50% of the activity of the zero value available.

The half-life test (i) described above usually gives the more accurate values; however, it is time consuming. If many substances are to be tested, one will because of the higher test speed, select the binding test (ii); he is too too suitable for screening, followed by a half-life test (i) for selected ones Connect connections. HBV core protein inhibitors in the sense of the inventions These are at least the half-life test described above (i) exist.

A particular embodiment of the invention relates to combinations of A) above dihydropyrimidines (I) and (Ia) and B) HBV polymerase inhibitors.

As HBV polymerase inhibitors B in the context of the invention, such substances referred to in the below-described endogenous polymerase assay of Ph. A. Furman et al. in Antimicrobial Agents and Chemotherapy, Vol. 36 (No. 2688 (1992), to inhibit the formation of an HBV DNA Double strands lead such that maximally 50% of the activity of the zero value result:

HBV virions from culture supernatants incorporate in vitro nucleoside 5'-triphosphates into the plus strand of HBV DNA. Using agarose gel electrophoresis, incorporation of [α- ³² P] deoxynucleoside 5'-triphosphate into the viral 3.2 kb DNA product in the presence and absence of a substance with potentially HBV-polymerase inhibitory properties is observed , HBV virions are obtained from the cell culture supernatant of HepG2.2.15 cells by precipitation with polyethylene glycol and concentrated. 1 volume of clarified cell culture supernatant is mixed with ¼ volume of an aqueous solution containing 50% by weight of polyethylene glycol 8000 and 0.6 M common salt. The virions are sedimented by centrifugation at 2,500 x g / 15 minutes. The sediments are resuspended in 2 ml of buffer containing 0.05 M Tris-HCl (pH 7.5) and dialyzed against the same buffer containing 100 mM potassium chloride. The samples can be frozen at -80 ° C. Each reaction batch (100 μl) contains at least 10 ⁵ HBV virions; 50 mM Tris-HCl (pH 7.5); 300mM potassium chloride; SOmM magnesium chloride; 0.1% ® Nonident P-40 (nonionic detergent from Boehringer Mannheim); 10 μM each dATP, dGTP and dTTP; 10 μCi [ ³² P] dCTP (3000 Ci / mmol, final concentration 33 nM) and 1 μM of the potential polymerase inhibitor in its triphosphorylated form. The samples are incubated at 37 ° C for one hour and then the reaction is stopped by addition of 50 mM EDTA. A 10% w / v SDS solution (containing 10 g of SDS per 90 ml of water) is added to a final concentration of 1% by volume (based on total volume) and proteinase K is added to a final concentration of 1 mg / ml added. After incubation at 37 ° C for one hour, samples are extracted with the same volume of phenol / chloroform / isoamyl alcohol (25: 24: 1 by volume), and from the aqueous phase the DNA is precipitated with ethanol. The DNA pellet is resuspended in 10 μl gel buffer (solution of 10.8 g Tris, 5.5 g boric acid and 0.75 g EDTA in 1 liter water (= TBE buffer)) and separated by electrophoresis in an agarose gel. The gel is either dried or the nucleic acids contained therein are transferred to a membrane by means of Southern transfer technology. Thereafter, the amount of gebil Deten and labeled DNA double strand in relation to the negative control (= Endo-Pol reaction without substance or with control substance without effect) be true. An HBV polymerase inhibitor is present when there is a maximum of 50% of the activity of the negative control.

Preferred HBV polymerase inhibitors B) include, for example
3TC = lamivudine logo CNRS logo INIST
= 4-amino-1 - [(2R-cis) -2- (hydroxymethyl) -1,3-oxathiolan-5-yl] -pyrimidin-2 (1H) -one, cf. EP-PS 382 526 (= US Pat. No. 5,047,407) and WO 91/11186 (= US Pat. No. 5,204,466);
Adefovir dipivoxil logo CNRS logo INIST
9- {2 - [[bis [(pivaloyloxy) methoxy] phosphinyl] methoxy] ethyl} adenine, cf. EP-PS 481 214 (= US-PS 5,663,159 and 5,792,756), US-PS 4,724,233 and 4,808,716;
BMS 200 475 =
[1S- (1.α, 3.α, 4.β)] -2-amino-1,9-dihydro-9- [4-hydroxy-3- (hydroxymethyl) -2-methylene-cyclopentyl] -6H-purine 6-on, cf. EP-PS 481 754 (= US Pat. Nos. 5,206,244 and 5,340,816), WO 98/09964 and 99/41275;
Abacavir logo CNRS logo INIST
(-) - (1S-cis) -4- [2-Amino-6- (cyclopropylamino) -9H-purin-9-yl] -2-cyclopentene-1-methanol, cf. EP-PS 349 242 (= US-PS 5 049 671) and EP-PS 434 450 (= US-PS 5 034 394);
FTC =
(2R-cis) -4-amino-5-fluoro-1- [2- (hydroxymethyl) -1,3-oxathiolan-5-yl] -pyrimidine-2 (1H) -one, cf. WO 92/14743 (= US Pat. No. 5,204,466, 5,210,085, 5,539,116, 5,700,937, 5,728,575, 5,814,639, 5,827,727, 5,852,027, 5,892,025, 5,914,331, 5) 914,400) and WO 92/18517;
β-L-FDDC =
5- (6-amino-2-fluoro-9H-purin-9-yl) -tetrahydro-2-furan-methanol, cf. WO 94/27616 (= US Pat. Nos. 5,627,160, 5,561,120, 5,631,239 and 5,830,881);
L-FMAU = 1- (2-deoxy-2-fluoro-β-L-arabinofuranosyl) -5-methylpyrimidine-2.4 (1H, 3H) -dione, cf. WO 99/05157, WO 99/05158 and US Pat. No. 5,753,789.

Another preferred embodiment of the invention relates to combinations of A) the above dihydropyrimidines (I) or (Ia) and B) lamivudine.  

Other preferred HBV antiviral agents B include e.g. B. Phenylpxopenamide the formula

wherein
R ¹ and R ² independently of one another are C ₁ -C ₄ -alkyl or together with the nitrogen atom to which they stand form a ring having 5 to 6 ring atoms which comprise carbon and / or oxygen,
R ³ -R ¹² independently of one another are hydrogen, halogen, C ₁ -C ₄ -alkyl, optionally substituted C ₁ -C ₄ -alkoxy, nitro, cyano or trifluoromethyl,
R ^{13 is} hydrogen, C ₁ -C ₄ -alkyl, C ₁ -C ₇ -acyl or aralkyl and
X is halogen or optionally substituted C ₁ -C ₄ -alkyl
mean,
and their salts.

These phenylpropenamides and processes for their preparation are known from WO 98/33501, to which reference is hereby made for the purpose of disclosure is taken. AT-61 is the compound of the above formula wherein X is chlorine, A 1- Piperidinyl and Y and Z are each phenyl. Own investigations have ge  shows that AT-61 is not an HBV core protein inhibitor for the purposes of this invention (Test above).

Another object of the invention is a method for producing the ge called combinations, after which the components A and B in suitable Combined or made way.

The present invention includes pharmaceutical preparations which, in addition to non- toxic, inert pharmaceutically acceptable excipients one or more inventions Contain the proper combinations or from a combi invention nation, as well as processes for the preparation of these preparations.

The quantitative ratio of components A and B of the combination according to the invention nations can vary within wide limits; preferably it is 5 to 500 mg A / 10 to 1000 mg B, especially 10 to 200 mg A / 20 to 400 mg B.

The combinations of the invention are intended in the above-mentioned pharma ceutical preparations generally in a concentration of about 0.1 to 99.5, preferably about 0.5 to 95 wt .-% of the total mixture be present.

The above-listed pharmaceutical preparations may except the Invention contain combinations according to other pharmaceutical active ingredients.

The preparation of the abovementioned pharmaceutical preparations can be in üb Licher way done by known methods, eg. B. by mixing the drug or the active ingredients with the carrier or excipients.

In general, it has been used in both human and veterinary medicine as proven advantageous combinations of the invention in total amounts of from about 0.5 to about 500, preferably 1 to 100 mg / kg of body weight per 24 hours, optionally in the form of several single doses to achieve the desired result  to administer. A single dose contains the active substance or active substances preferably in amounts of from about 1 to about 80, especially 1 to 30 mg / kg of body mass. However, it may be necessary to depend on the dosages mentioned soft, depending on the type and body weight of the behan object, the nature and severity of the disease, the method of preparation and the application of the drug as well as the period or interval within which the administration takes place.

The indications for the combinations according to the invention include:

• 1. the treatment of acute and chronic viral infections leading to one can cause infectious hepatitis, preferably the treatment of acute and chronic hepatitis B virus infections;
• 2. the treatment of acute and chronic HBV infections in Koin infection with the hepatitis delta virus;
• 3. the treatment of acute and chronic HBV infections in Koin infection with other viruses, such as HIV or HCV, and
• 4. the prophylactic / therapeutic treatment in transplants, such as z. B. liver transplants.

Another object of the invention are therefore the combinations defined above for use as a medicine.

Another object of the invention are pharmaceutical compositions containing at least one the above defined combinations and optionally further pharmaceutical Agents.  

Another object of the invention is a process for the preparation of Medicaments, according to which at least one of the combinations defined above, optionally using customary excipients and carriers, in one transferred suitable application form.

Another object of the invention is the use of the above defined Combinations for the manufacture of a medicament.

Another object of the invention is the use of the above defined Combinations for the manufacture of a medicament for the treatment and prophylaxis the diseases described above, preferably viral diseases, especially hepatitis B.

Composition (mg / l) of the 1x medium prepared from ®seromed RPMI 1640:

NaCl 6000 KCl 400 Na ₂ HPO ₄ × 7 H ₂ O 1512 MgSO ₄ .7H ₂ O 100 Ca (NO ₃ ) ₂ × 4 H ₂ O 100 D-glucose 2000 phenol 5 NaHCO ₃ 2000 phenol 5 NaHCO ₃ 2000

L-arginine 200 L-asparagine 50 L-aspartic acid 20 L-cystine 50 L-glutamine 300 L-glutamic acid 20 glycine 10 L-histidine 15 L-hydroxyproline 20 L-isoleucine 50 L-leucine 50 L-lysine × HCl 40 L-methionine 15 L-phenylalanine 15 L-proline 20 L-serine 30 L-threonine 20 L-tryptophan 5 L-tyrosine 20 L-valine 20

glutathione 1 biotin 0.2 Vitamin B ₁₂ 0.005 D-Ca pantothenate 0.25 choline chloride 3 folic acid 1 i-inositol 35 nicotinamide 1 p-aminobenzoic acid 1 Pyridoxine × HCl 1 riboflavin 0.2 Thiamine × HCl 1

Composition (mg / l) of the 1x medium prepared from ®seromed Medium 199 with Earle's salts:

NaCl 6800 KCl 400 NaH ₂ PO ₄ × H ₂ O 140 MgSO ₄ .7H ₂ O 200 CaCl ₂ 200 D-glucose 1000 phenol 17 NaHCO ₃ 2200

DL-alanine 50 L-arginine × HCl 70 DL-aspartic acid 60 L-cysteine × HCl 0.1 L-cystine 20 L-glutamine 100 DL-glutamic acid × H ₂ O 150 AL = L <glycine L-histidine × HCl 50 L-hydroxyproline 20 DL-isoleucine 10 DL-leucine 40 L-lysine × HCl 120 DL-methionine 70 DL-phenylalanine 30 L-proline 50 DL-serine 40 DL-threonine 50 DL-tryptophan 60 L-tyrosine 20 DL-Valine 40 glutathione 50 sodium 0.05 Fe (NO ₃ ) ₃ 50 ®Tween 80 * 0.1 20 adenine sulfate 10 Guanine × HCl 0.3 hypoxanthine 0.3 thymine 0.3 uracil 0.3 xanthine 0.3 * Brand of Atlas Chemistry

ATP, Na ₂ 1 AMP 0.2 ascorbic acid 0.05 biotin 0.01 calciferol 0.1 D-Ca pantothenate 0.01 choline chloride 0.5 folic acid 0.01 i-inositol 0.05 menadione 0.01 nicotinic acid 0,025 nicotinamide 0,025 p-aminobenzoic acid 0.05 Pyridoxal × HCl 0,025 Pyridoxine × HCl 0,025 riboflavin 0.01 Thiamine × HCl 0.01 DL-a-tocopherol phosphate-Na ₂ 0.01 Vitamin A 0.1 cholesterol 0.2 2-deoxy-D-ribose 0.5 D-ribose 0.5

Medium composition (mg / l) of the MEM-lx solution prepared from Gibco 51091-015:

CaCl ₂ × 2 H ₂ O 264 KCl 400 MgSO ₄ .7H ₂ O 200 NaCl 6800 NaHCO ₃ 2200 NaH ₂ PO ₄ × 2 H ₂ O 158 phenol 10

L-histidine × HCl × H ₂ O 41.92 L-isoleucine 52.46 L-lysine × HCl 73.06 L-phenylalanine 33.02 L-threonine 47.64 L-tryptophan 10.20 L-tyrosine 36.22 L-valine 46.86 D-Ca pantothenate 1.00 choline chloride 1.00 folic acid 1.00 nicotinamide 1.00 Pyridoxal × HCl 1.00 riboflavin 0.10 Thiamine × HCl 1.00

Examples HBV in cell culture

The antiviral effect of the combinations according to the invention was based on to M. A. Seils et al., Proc. Natl. Acad. Sci. 84, 1005-1009 (1987) and B.E. Korba et al., Antiviral Research 19, 55-70 (1992) carried out.

The tests of the combinatorial test of the test substances were carried out by means of chess board titration (checkerboard titration).

The antiviral tests were carried out in 96 plates of microtiter plates. The first vertical row of the plate received only HepG2.2.15 cells in growth medium. you served as a virus control.

Stock solutions of the test compounds (50 mM) were first dissolved in DM50; further dilutions were made in growth medium. The remaining bowls contained the combinations according to the invention or their individual components in the test concentrations of z. B. 5 uM to 0.01 uM, starting from A2 to H11 of 96-because microtiter plate.

Stock solutions of the substances to be tested were on separate 96-well plates prepared and then put together on the test plate with HepG2.2.15 cells Pipette. This resulted in test concentrations in the range of approx. 10-50 times higher. and covered below the IC-50 concentrations.

The assay was incubated for 8 days at 37 ° C and 5% CO ₂ (v / v). On day 4, the medium was replaced with fresh inhibitor-containing medium.

cytotoxicity

Before harvesting the supernatants / cell lysates to determine the antiviral effect HepG2.2.15 cells were visualized by light microscopy or by biochemical analysis white method (eg Alamar Blue stain or Trypan blue stain) on cytotoxin examined toxic changes.

Substance-induced cytotoxic or cytostatic changes in HepG2.2.15 Cells were z. B. light microscopy as changes in cell morphology he averages. Such substance-induced changes in HepG2.2.15 cells in the Compared to untreated cells were z. B. as cell lysis, vacuolation or altered cell morphology visible. 50% cytotoxicity ("Tox.-50") mean that 50% of the cells have a similar morphology to the corresponding cell control respectively.

The compatibility of some combinations of the invention was additionally on other host cells, such. B. HeLa cells, primary peripheral blood cells of the Humans or transformed cell lines, such as H-9 cells, have been tested.

There could be no cell-cytotoxic changes in the test concentration range be determined.

Determination of antiviral activity

Subsequently, the supernatants / cell lysates were harvested and reduced Pressure on nylon membrane covered 96-well dot blot chambers (ent speaking the manufacturer's instructions) sucked.

In brief: After transfer of the supernatants or whole cell lysates to the nylon Membrane of the blotting apparatus (see above) were the nucleic acids contained therein denatured (1.5 M NaCh 0.5 N NaOH), neutralized (3 M NaCh 0.5 M Tris HCl,  pH 7.5) and washed (2 × SSC). Subsequently, the DNA was incubated the filter baked at 120 ° C, 2-4 hours, to the membrane.

Hybridization of the DNA

The detection of viral DNA from the treated HepG2.2.15 cells on the Nylon filters were usually labeled with nonradioactive, digoxigenin-labeled ones Hepatitis B-specific DNA probes are performed, each according to manufacturer labeled with digoxigenin, purified and used for Hybidisierung were.

Briefly, prebybidation and hybridization were performed in 5x SSC, 1X Blocking reagent, 0.1% N-lauroyl sarcosine, 0.02% SDS and 100 μg sperm DNA of the herring. The prehybridization was carried out for 30 minutes at 60 ° C, the speci hybridization with 20-40 ng / ml of digoxigenized, denatured HBV specific DNA (14 hours, 60 ° C). Subsequently, the filters were washed.

Detection of HBV DNA by digoxigenin antibody

The immunological detection of the digoxigenin-labeled DNA was carried out after Manufacturer's instructions.

Briefly, the filters were washed and placed in a blocking reagent (after Manufacturer's specification) prehybridized. Subsequently, with an anti-DIG anti body, which was coupled with alkaline phosphatase, hybridized for 30 minutes. To In a washing step, the alkaline phosphatase substrate, CSPD, was added Add, incubated with the filters for 5 minutes, then wrapped in plastic wrap and incubated for a further 15 minutes at 37 ° C. Chemiluminescence of Hepatitis B Specific DNA signals were via exposure of the filters using biolumin on an x-ray film or with a lumi-imager  (Incubation depending on signal strength: about 2 minutes to about 2 hours) and the degree of Measure blackness.

The inhibitory values were determined according to the cut-off values from the internal test Controls converted to% inhibition values. To analyze the synergistic effect The combinations of the combinations were the difference values of calculated and ge measured inhibition values of each combination; see. Prichard et al., Antimicrob. Agents Chemother. 37, 540-545 (1993).

The treatment of HBV with the combinations of the invention has anti viral better than the single treatment; the treatment of hepatitis B virus produ decorating HepG2.2.15 cells with the combinations according to the invention led to a greater reduction of intracellular viral DNA; the combinations Treatment is synergistically effective.

Claims (16)

1. Combinations of at least one dihydropyrimidine and B) at least one of A different HBV antiviral agent. 2. Combinations according to claim 1, wherein the dihydropyrimidine A of the formula
or their mesomeric form
wherein
R ^{1 is} phenyl, furyl, thienyl, triazolyl, pyridyl, cycloalkyl having 3 to 6 carbon atoms or radicals of the formulas
or
where the ring systems listed above are optionally mono- or polysubstituted by identical or different substituents selected from the group consisting of halogen, trifluoromethyl, nitro, cyano, trifluoromethoxy, carboxyl, hydroxyl, (C ₁ -C ₆ ) -alkoxy, (C ₁ - C ₆ ) - alkoxycarbonyl and (C ₁ -C ₆ ) alkyl, wherein the alkyl radical may in turn be substituted by aryl having 6 to 10 carbon atoms or halogen, and the ring systems listed optionally by
-SR ⁶ , -NR ⁷ R ⁸ , -CO-NR ⁹ R ¹⁰ , -SO ₂ -CF ₃ and -A-CH ₂ -R ^{11 are} substituted,
wherein
R ^{6 is} optionally halogen-substituted phenyl,
R ⁷ to R ¹⁰ independently of one another are hydrogen, phenyl, hydroxyl-substituted phenyl, hydroxy, (C ₁ -C ₆ ) -acyl or (C ₁ -C ₆ ) -alkyl, where the alkyl radical is in turn replaced by hydroxy, (C ₁ -C ₆ ) - alkoxycarbonyl, phenyl or hydroxy substituted phenyl may be substituted,
A is a radical -O-, -S-, -SO- or -SO ₂ -,
R ^{11 is} phenyl which is optionally mono- to polysubstituted by identical or different substituents selected from the group halogen, nitro, trifluoromethyl, (C ₁ -C ₆ ) alkyl and (C ₁ -C ₆ ) - alkoxy substituted , mean
R ^{2 is} a radical of the formulas -XR ¹² or -NR ¹³ R ¹⁴ ,
wherein
X is a single bond or oxygen,
R ^{12 is} hydrogen, straight-chain or branched (C ₁ -C ₆ ) -alkoxy-carbonyl, a straight-chain, branched or cyclic, saturated or unsaturated (C ₁ -C ₈ ) -hydrocarbon radical which may contain one or two identical or different heterokain members from the group -O-, -CO-, -NH-, -N- (C ₁ -C ₄ alkyl) -, -S- or -SO ₂ - and optionally substituted by halogen, nitro, cyano, hydroxy, aryl with 6 to 10 carbon atoms, aralkyl having 6 to 10 carbon atoms, heteroaryl or a group of the formula
-NR ¹⁵ R ^{16 is} substituted,
in which R ¹⁵ and R ¹⁶ independently of one another are hydrogen, benzyl or (C ₁ -C ₆ ) -alkyl,
R ¹³ and R ¹⁴ independently of one another are hydrogen, (C ₁ -C ₆ ) -alkyl or cycloalkyl having 3 to 6 carbon atoms,
R ^{3 is} hydrogen, amino or a radical of the formula
or
Formyl, cyano, hydroxy-substituted (C ₁ -C ₆ ) -alkylthio, trifluoromethyl or pyridyl or
a straight-chain, branched or cyclic, saturated or unsaturated hydrocarbon radical having up to 8 carbon atoms, optionally mono- or polysubstituted, identically or differently, by aryloxy having 6 to 10 carbon atoms, azido, halogen, cyano, hydroxyl, carboxyl, (C ₁ -C ₆ ) alkoxycarbonyl, a 5- to 7-membered heterocyclic ring, (C ₁ -C ₆ ) -alkylthio or (C ₁ -C ₆ ) -alkoxy (where the alkylthio or alkoxy radical in turn is substituted by azido, amino, hydroxyl and / or substituted by the group - (CO) _a -NR ¹⁷ R ¹⁸ ,
where a is zero or 1,
R ¹⁷ and R ^{18 are} independently hydrogen or aryl, aralkyl of 6 to 10 carbon atoms or (C ₁ -C ₆ ) alkyl, optionally substituted by (C ₁ -C ₆ ) alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl are, where phenyl and benzyl optionally substituted one or more times, identically or differently, by hydroxy, carboxyl, (C ₁ -C ₆₎ -alkyl or (C ₁ - C ₆₎ -alkoxy and / or (C ₁ -C ₆ ) -Alkyl optionally substituted by -NH-CO-CH ₃ or -NH-CO-CF ₃ substituted,
or
R ¹⁷ and R ^18, together with the nitrogen atom to which they stand, signify a morpholinyl, piperidinyl or pyrrolidinyl ring,
or
R ^{3 is} optionally methoxy-substituted phenyl
or
R ² and R ³ together have a radical of the formula
R ^{4 is} hydrogen, (C ₁ -C ₄ ) -alkyl, (C ₂ -C ₄ ) -alkenyl, benzoyl or acyl having 2 to 6 carbon atoms, preferably hydrogen, methyl, benzoyl or (C ₂ -C ₆ ) -acyl, and
R ^{5 is} pyridyl, pyrimidyl or pyrazinyl, each up to 3-fold, identically or differently, by halogen, hydroxy, cyano, trifluoromethyl, (C ₁ -C ₆ ) -alkoxy, (C ₁ -C ₆ ) -alkyl, (C ₁ -C ₆₎ -alkylthio, carbalkoxy, (C ₁ - C ₆₎ acyloxy, amino, nitro, mono- or di- (C ₁ -C ₆₎ alkylamino may be stituiert sub,
mean,
and their salts. 3. Combinations according to claim 1, wherein the dihydropyrimidine A of the formula
or their isomeric form
corresponds to, in which
R ^{1 is} phenyl, furyl, thienyl, pyridyl, cycloalkyl having 3 to 6 carbon atoms or a radical of the formulas
where the ring systems listed above are optionally mono- or polysubstituted by identical or different substituents selected from the group consisting of halogen, trifluoromethyl, nitro, cyano, trifluoromethoxy, carboxyl, hydroxyl, (C ₁ -C ₆ ) -alkoxy, (C ₁ - C ₆ ) - alkoxycarbonyl and (C ₁ -C ₆ ) -alkyl, where the alkyl radical may in turn be substituted by aryl having 6 to 10 carbon atoms or halogen,
and / or the ring systems listed optionally by groups of the formulas -SR ⁶ , -NR ⁷ R ⁸ , -CO-NR ⁹ R ¹⁰ ,
-SO ₂ -CF ₃ and -A-CH ₂ -R ^{11 are} substituted,
wherein
R ^{6 is} optionally halogen-substituted phenyl,
R ⁷ to R ¹⁰ independently of one another are hydrogen, phenyl, hydroxyl-substituted phenyl, hydroxy, (C ₁ -C ₆ ) -acyl or (C ₁ -C ₆ ) -alkyl, where the alkyl radical is in turn replaced by hydroxy, (C ₁ -C ₆ ) - alkoxycarbonyl, phenyl or hydroxy substituted phenyl may be substituted,
A is a radical -O-, -S-, -SO- or -SO ₂ -,
R ^{11 is} phenyl which is optionally mono- to polysubstituted by identical or different substituents selected from the group halogen, nitro, trifluoromethyl, (C ₁ -C ₆ ) alkyl and (C ₁ -C ₆ ) - alkoxy substituted , mean
R ^{2 is} a radical of the formulas -OR ¹² or -NR ¹³ R ¹⁴
wherein
R ^{12 is} hydrogen, (C ₁ -C ₆ ) -alkoxycarbonyl or a straight-chain, branched or cyclic, saturated or unsaturated (C ₁ -C ₈ ) -hydrocarbon radical which optionally has one or two identical or different heterokain members from the group -O -
-CO-, -NH-, -N- (C ₁ -C ₄ alkyl) -, -S- and -SO ₂ - and optionally substituted by halogen, nitro, cyano, hydroxy, aryl having 6 to 10 carbon atoms or Aralkyl having 6 to 10 carbon atoms, heteroaryl or a group of the formula -NR ¹⁵ R ^{16 is} substituted,
in which R ¹⁵ and R ¹⁶ independently of one another are hydrogen, benzyl or (C ₁ -C ₆ ) -alkyl,
R ¹³ and R ¹⁴ independently of one another are hydrogen, (C ₁ -C ₆ ) -alkyl or cycloalkyl having 3 to 6 carbon atoms,
R ^{3 is} hydrogen, amino or a radical of the formula
or
Formyl, cyano, hydroxy substituted (C ₁ -C ₄ ) alkylthio, tri fluoromethyl or
a straight-chain, branched or cyclic, saturated or unsaturated hydrocarbon radical having up to 8 carbon atoms, optionally mono- or polysubstituted, identically or differently, by aryloxy having 6 to 10 carbon atoms, azido, cyano, hydroxyl, carboxyl, (C ₁ -C ₆ ) Alkoxycarbonyl, a 5- to 7-membered heterocyclic ring, (C ₁ -C ₆ ) -alkylthio or (C ₁ -C ₆ ) -alkoxy (where the alkylthio or alkoxy radical in turn may be substituted by azido, amino or hydroxyl can) and / or is substituted by the group - (CO) _a - NR ¹⁷ R ¹⁸ ,
where a is zero or 1,
R ¹⁷ and R ^{18 are} independently hydrogen or aryl, aralkyl of 6 to 10 carbon atoms or (C ₁ -C ₆ ) alkyl, optionally substituted by (C ₁ -C ₆ ) alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl are, where phenyl and benzyl optionally substituted one or more times, identically or differently, by hydroxy, carboxyl, (C ₁ -C ₆₎ -alkyl or (C ₁ - C ₆₎ -alkoxy and / or (C ₁ -C ₆ ) -Alkyl optionally substituted by -NH-CO-CH ₃ or -NH-CO-CF ₃ ,
or
R ¹⁷ and R ^18, together with the nitrogen atom to which they stand, signify a morpholinyl, piperidinyl or pyrrolidinyl ring,
R ^{4 is} hydrogen, (C ₁ -C ₄ ) -alkyl, acetyl or benzoyl,
D is an oxygen or sulfur atom and
R ^{5 is} hydrogen, halogen or straight-chain or branched alkyl having up to 6 carbon atoms
mean,
and their salts. 4. Combinations according to claims 1 and 2, wherein the dihydropyrimidine A is the formulas
and / or their mesomeric forms and / or their salts. 5. Combinations A) of at least one HBV-core protein inhibitor and B) at least one of A different HBV antiviral agent. 6. Combinations according to claims 1 to 5, wherein component B is an HBV Polymerase inhibitor is. 7. Combinations according to claims 1 to S. wherein component B lamivudine is.   8. Combinations according to claims 1 to 5, wherein component B from the compounds of formula
wherein
R ¹ and R ² independently of one another are C ₁ -C ₄ -alkyl or together with the nitrogen atom to which they stand form a ring having 5 to 6 ring atoms which comprise carbon and / or oxygen,
R ³ -R ^{12 are} independently hydrogen, halogen, C ₁ -C ₄ -alkyl, optionally substituted C ₁ -C ₄ -alkoxy, nitro, cyano or trifluoromethyl,
R ^{13 is} hydrogen, C ₁ -C ₄ -alkyl, C ₁ -C ₇ -acyl or aralkyl and
X is halogen or optionally substituted C ₁ -C ₄ -alkyl
mean,
and their salts are selected. 9. Combinations according to claim 8, wherein
X is chlorine, A is 1-piperidinyl and Y and Z are each phenyl. 10. A process for the preparation of the combinations according to claims 1 to 9, characterized in that the components A and B in suitable Combined or made way. 11. Combinations according to claims 1 to 9 for use as medicaments. 12. A pharmaceutical composition containing at least one combination according to claims 1 to 9 and optionally other active pharmaceutical ingredients. 13. A process for the preparation of medicaments according to claim 12, which comprises at least one combination according to claims 1 to 9, optionally using conventional excipients and carriers, into a suitable Transferred application form. 14. Use of combinations of claims 1 to 9 for the preparation of a Drug. 15. Use of combinations of claims 1 to 9 for the preparation of a Medicament for the treatment and prophylaxis of viral diseases. 16. Use of combinations of claims 1 to 9 for the preparation of a Medicament for the treatment and prophylaxis of hepatitis B.