WO2001045712A1 - Combinations of medicaments for treating viral diseases - Google Patents

Abstract

The invention relates to combinations of dihydropyrimidines, lamivudin and, optionally, interferon which inhibit the multiplication of HBV viruses, better than conventional agents.

Description

Drug combinations against viral diseases

The invention relates to combinations of A) non-nucleoside inhibitors, e.g. Dihydropyrimidines, B) other antiviral agents and optionally C)

Immunomodulators, a process for their preparation and their use as medicines, in particular for the treatment and prophylaxis of HBV infections.

“Combinations” in the sense of the invention are understood not only to be dosage forms which contain all components (so-called fixed combinations) and combination packs which contain the components separately from one another, but also components which are applied simultaneously or at different times, provided that they are used for treatment or prophylaxis of the same disease.

The hepatitis B virus belongs to the Hepadna virus family. It causes an acute and / or a persistent-progressive, chronic illness. Various other clinical manifestations in the clinical picture are also caused by the hepatitis B virus - in particular chronic liver inflammation, cirrhosis and hepatocellular carcinoma. Furthermore, co-infection with the hepatitis delta virus can have a negative impact on the course of the disease.

Several options have already been proposed for virus inhibition:

1. the inhibition of the polymerase of HBV by analogues of the substrates of this enzyme such as lamivudine, FTC, adefovir dipivoxil, abacavir, ß-L-FDDC,

L-FMAU and BMS 200475:

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

Lamivudine has recently been used to treat patients with chronic HBV infection. However, it shows a high rebound effect after stopping the therapy and leads to resistance during long-term therapy. For Adefovir Dipivoxil, BMS 200475 and the other inhibitors mentioned above, there is as yet no generally accessible clinical experience.

2. the inhibition of HBV by immunological principles such as the

Treatment of chronic hepatitis by interferon; however, it is only moderately effective and has undesirable side effects. Combinations of interferon with lamivudine are not synergistically effective.

The stimulation of host immune defense, e.g. with thymosin-α, has already been proposed.

3. inhibition by other active substances, the mode of action of which is not known or is the subject of speculation, such as AT-61 = N - [(1E) - 2-chloro-2-phenyl-l- (l-piperidinylcarbonyl) ethenyl] benzamide, which apparently interferes with the process of packaging the pregenomic RNA into the unfinished core particles; see. King et al., Antimicrob. Agents and chemother. 42, 3179-3186 (1998). Even if the mechanism has not yet been fully elucidated, the authors seem to rule out an effect on the core protein. The in vitro combination of AT-61 and lamivudine is described as being synergistically effective.

Previous therapeutic agents for the treatment of HBV-infected patients, such as interferon or lamivudine, are used as monotherapy. It is known from clinical studies that combinations of both inhibitors have no advantage in combating HB V diseases. New means for better and more effective therapy are therefore desirable.

It has now surprisingly been found that combinations of A) non-nucleoside inhibitors such as dihydropyrimidines, B) other HBV antivirals

Agents and, where appropriate, C) immunomodulators do not have the disadvantages of the prior art, or only partially.

The invention therefore relates to combinations A) of at least one dihydropyrimidine, B) at least one HBV-antiviral agent different from A, preferably an HBV polymerase inhibitor, and optionally C) at least one immunomodulator. The invention thus relates to combinations of nucleoside and non-nucleoside inhibitors and, if appropriate, immunomodulators for the treatment and prophylaxis of HBV infections and the use of these combinations for the treatment of HB V-induced diseases.

The combinations according to the invention unpredictably inhibit the multiplication of the HBV virus significantly better than the agents known from the prior art or their known combinations. The use of the combinations according to the invention offers valuable treatments for HBV-induced diseases

Advantages compared to monotherapy with the individual compounds, namely mainly a synergistic antiviral activity, but also a good tolerance 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 individual components.

Preferred dihydropyrimidines A correspond, for example, to the formula

or their isomeric form

Wonn

R phenyl, furyl, thienyl, triazolyl, pyridyl, cycloalkyl having 3 to 6 carbon atoms or radicals of the formulas

or

means, where the ring systems listed above, if appropriate, once or several times, identically or differently, by substituents selected from the group halogen, trifluoromethyl, nitro, cyano, trifluoromethoxy, carboxyl, hydroxyl, -C _ö alkoxy, d-Ce-alkoxycarbonyl and -C _ö alkyl, are substituted, the alkyl radical in turn being substituted by aryl having 6 to 10 carbon atoms or halogen,

and the ring systems listed, if appropriate

-SR ⁶ , -NR ⁷ R ⁸ , -CO-NR ⁹ R ¹⁰ , -SO ₂ -CF ₃ and -A-CHrR ^{11 are} substituted, wherein

R ⁶ optionally halogen-substituted phenyl,

R to R are independently hydrogen, phenyl, hydroxy-substituted phenyl, hydroxy, QC _ö acyl or Ci-Cβ-alkyl, where the alkyl radical for its part substituted hydroxy-substituted by hydroxy, dC _ö alkoxycarbonyl, phenyl or phenyl may be substituted .

A is a radical -O-, -S-, -SO- or -SO ₂ -,

, R is substituted ^and phenyl which is optionally monosubstituted to polysubstituted by identical or different substituents selected from the group halogen, nitro, trifluoromethyl, QC _ö alkyl and Cι-C ₆ -alkoxy, mean

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 CrC _δ alkoxycarbonyl, a straight-chain, branched or cyclic, saturated or unsaturated C _j -C ₈ hydrocarbon radical, which may be a or two identical or different hetero chain links from the group -O-, -CO-, -NH-, -N- (dC ₄ -alkyl) -,

-S- or -SO ₂ - and which is optionally substituted by halogen, nitro, cyano, hydroxy, aryl having 6 to 10 carbon atoms, aralkyl having 6 to 10 carbon atoms, heteroaryl or a group of the formula -NR ¹⁵ R ¹⁶ ,

in which R ¹⁵ and R ¹⁶ independently of one another are hydrogen, benzyl or dC 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

H ₃ CO

OCH, or

Formyl, cyano, hydroxy-substituted -CC ₆ alkylthio, trifluoromethyl or pyridyl or

a straight-chain, branched or cyclic, saturated or unsaturated hydrocarbon radical having up to 8 carbon atoms, optionally one 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, dC ₆ - alkylthio or dC ₆ alkoxy (the alkylthio or alkoxy radical being its - may be substituted by azido, amino, hydroxyl) and / or 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 having 6 to 10 carbon atoms, aralkyl having 6 to 10 carbon atoms or C ₁ -C ₆ -alkyl, which are optionally substituted by -CC ₆ -alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl wherein phenyl and benzyl are optionally mono- or polysubstituted, identically or differently by hydroxyl, carboxyl, _Cι-C6 alkyl or Cι-C substituted ₆ alkoxy and / or _Cι-C6 alkyl optionally substituted by -NH-CO- CH ₃ or -NH-CO-CF _{3 is} substituted,

or

R ¹⁷ and R ¹⁸ together with the nitrogen atom on which they stand mean a morpholinyl, piperidinyl or pyrrolidinyl ring,

or

R optionally methoxy-substituted phenyl

or

R and R together form a radical of the formula

R 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 pyridyl, pyrimidyl or pyrazinyl, each up to 3 times, the same or different by halogen, hydroxy, cyano, trifluoromethyl, Ci-C _ö alkoxy, Ci-C ₆ alkyl, dC ₆ alkylthio, carbalkoxy, dC ₆ -acyloxy, amino, nitro, mono- or di-C ₆ -C ₆ -alkylamino can be substituted,

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 (la) are in tautomeric equilibrium:

( there)

Preferred compounds of the formulas (I) and (Ia) are those in which

R ^{1 is} phenyl, furyl, thienyl, triazolyl, pyridyl, cyclopentyl, cyclohexyl or

Remains of the formulas

or

means, where the above-mentioned ring systems optionally one or two, identical or different by substituents selected from the group halogen, trifluoromethyl, nitro, -SO ₂ -CF ₃ , methyl, cyano, trifluoromethoxy, hydroxy, carboxyl, methoxycarbonyl or Residues 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,

-O-CH ₂ -C ₆ H ₅ or -S-pCl-C ₆ B ι are substituted,

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 ₄ -alkenyl, dC -alkoxycarbonyl or dC ₄ -alkyl, where the alkyl radical is optionally substituted by pyridyl, cyano, phenoxy, benzyl or by a radical of the formula -NR ¹⁵ R ¹⁶ , in which

R ⁵ and R ^{6 are} independently hydrogen, benzyl or -CC ₄ alkyl,

R ¹³ and R ¹⁴ independently of one another denote hydrogen, -CC alkyl or cyclopropyl, R ^{3 is} hydrogen, amino or a radical of the formula

H ₃ CO

OCH,

or

Formyl, cyano, hydroxy-substituted C ₁ -C ₄ alkylthio, trifluoromethyl, cyclopropyl, pyridyl or C -C alkyl means, where the alkyl radical optionally by halogen, dC ₄ alkoxycarbonyl, hydroxy and / or by 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 Ci

C ₄ alkyl, which are optionally substituted by C ₃ -C ₃ -alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl, phenyl and benzyl independently of one another optionally optionally one or two times, identically or differently, by hydroxyl, carboxy, Ci-d- Alkyl or -CC ₃ alkoxy are substituted and dC ₃ alkyl is 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 on which they stand mean a morpholinyl, piperidinyl or pyrrolidinyl ring,

or

optionally methoxy substituted phenyl or

R and together a remainder of the formula

° ^

R> hydrogen, methyl, vinyl or acetyl and

R ^{5 is} pyridyl, pyrimidyl or pyrazinyl, and their salts.

Compounds of the formulas (I) and (Ia) in which

R ^{1 is} phenyl, furyl, thienyl, triazolyl, pyridyl, cyclopentyl, cyclohexyl or

Remains of the formulas

or

means, the above-mentioned ring systems optionally up to two, identical or different by substituents selected from the group fluorine, chlorine, bromine, iodine, hydroxy, trifluoromethyl, nitro, -SO ₂ -CF ₃ , Methyl, cyano, trifluoromethoxy, carboxyl, methoxycarbonyl or residues 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-OHj-C _ö E, -O-CH ₂ -C ₆ H ₅ or -S-pCl- CβΑi are substituted,

R ^{2 is} a radical of the formulas -XR ¹² or -NR ¹³ R ¹⁴ , in which

X is a single bond or an oxygen atom,

R ^{12 is} hydrogen, C ₂ -C ₃ alkenyl, dC ₄ alkoxycarbonyl or dC ₄ alkyl, in which the alkyl radical is 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 are hydrogen or methyl,

R ¹³ and R ¹⁴ independently of one another are hydrogen, C ₃ -C ₃ -alkyl or cyclopropyl,

R ^{3 is} hydrogen, amino or a radical of the formula

H ₃ CO

OCH ₃ or

Formyl, cyano, trifluoromethyl, cyclopropyl, pyridyl or dC ^ alkyl means, where the alkyl radical is optionally substituted by fluorine, chlorine, -C-C ₃ - alkoxycarbonyl or hydroxy, or R ³ optionally methoxy-substituted phenyl or

R ² and R ³ together represent a radical of the formula

R ^{4 is} hydrogen, methyl, vinyl or acetyl and

R ^{5 is} pyridyl, pyrimidyl or pyrazinyl, and their salts.

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 represent a radical of the formula

R ^{4 is} hydrogen, vinyl or acetyl and

R ^{5 is} pyridyl. Particularly preferred dihydropyrimidines A are listed in Table A.

Table A:

The dihydropyrimidines A listed in Table B are very particularly preferred.

Table B:

Particularly preferred dihydropyrimidines A are the above compounds 24, 25 and 41 to 44 of Table B. The following compounds are very particularly preferred:

their isomeric forms and their salts.

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

[A] aldehydes of the formula

R ^ CHO (II)

where R has the meaning given above,

with arnidines of the formula

where R • 5 has the meaning given above,

and compounds of the formula

R ³ -CO-CH ₂ -CO-R ² (TV)

in which R ² and R ^{3 have} the meanings given above,

with or without addition of base or acid, if appropriate in the presence of inert organic solvents, or

[B] Compounds of the formula

wherein R> 1 - bui.s, r R> 3 have the meanings given above,

with amidines of the formula

where R has the meaning given above, with or without addition of base or acid, if appropriate in the presence of inert organic solvents, preferably at temperatures from 20 to 150 ° C., or

[C] Aldehydes of the formula

R ^ CHO (II)

where R ^{1 has} the meaning given above,

with compounds of the formula

R - C = C - CO-R ² (VI)

H

NU,

wherein R and R have the meanings given above,

and amidines of the formula (III) as described above or

[D] aldehydes of the formula (IT) with compounds of the formula (IV) and imino ethers of the formula

wherein R ^{5 has} the meaning given above and R ^{1 represents} (dC ₄ ) alkyl,

reacted in the presence of ammonium salts.

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

NaOAc

All inert organic solvents are suitable as solvents for process variants A, B, C and D. These preferably include 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

Hexamethylphosphoramide.

The reaction temperatures can be varied within a wide range. In general, the process is carried out in the range from 20 to 150 ° C., but preferably at the boiling point of the particular solvent.

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

The reaction can be carried out with or without addition of base or acid; the presence of weaker acids such as acetic acid or formic acid is preferred. The aldehydes (II) used as starting materials are known or can be prepared by methods known from the literature [cf. TD Harris and GP Roth, J. Org. Chem. 44, 146 (1979), DE-OS 2 165 260 and 2 401 665, Mijano et al., Chem. Abstr. 59, (1963), 13 929 c, 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 (TV) used as starting materials are known or can be prepared by methods known from the literature [e.g. D. 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 be prepared by methods known from the literature [cf. G. Jones, "The Knoevenagel Condensation" in Organic Reactions, Vol. XV, 204 ff. (1967)].

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

The compounds (E) can be prepared by using compounds of the formula

R ⁵ -CN (VIII)

where R ^{5 has} the meaning given above, as usual via the imino ethers and finally reacted 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 from the literature, such as, for example, 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.

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

5

R -H (IX)

5 in which the hydrogen is in the ortho position to the nitrogen and R has the meaning given above,

first preferably at 50 to 150 ° C., in particular at about 100 ° C., in H ₂ O / glacial acetic acid to give the corresponding N-oxides and then with trimethylsilyl cyanide (TMSCN) according to processes known from the literature, for example in the inert types listed above Solvents, preferably acetonitrile, THF, toluene at room temperature or at reflux temperature, optionally with the addition of bases such as triethylamine or DBU, or by reacting in compounds of the formula

where 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 event that R denotes difluoropyridyl, compounds of the formula

where Y 'and Z' independently of one another are chlorine or bromine,

with alkali metal or ammonium fluorides, preferably potassium fluoride, using processes known from the literature, if appropriate in polar solvents, such as, for example, polyglycols, polyglycol ethers, DMSO or sulfolane, optionally with the addition of phase transfer catalysts, in the sense of a halogen / fluorine exchange reaction.

The above procedure is exemplified for the 3,5-difluoropyridyl compounds by the following reaction scheme:

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

Further preferred dihydropyrimidines A correspond to the formula

or their isomeric form

wherein

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

means, where the ring systems listed above, if appropriate, once or several times, identically or differently, by substituents selected from the group halogen, trifluoromethyl, nitro, cyano, trifluoromethoxy,

Carboxyl, hydroxyl, -CC ₆ alkoxy, d-Cβ-alkoxycarbonyl and Ci-Cβ-alkyl, are substituted, the alkyl radical in turn by aryl with 6 to 10th

Carbon atoms or halogen can be substituted, and / or the listed ring systems optionally by groups of

Formulas -SR ⁶ , -NR ⁷ R ⁸ , -CO-NR ⁹ R ¹⁰ ,

-SO ₂ -CF ₃ and -A-CH ₂ -R ^{π are} substituted, wherein

R ₁ 6 optionally halogen-substituted phenyl,

R ⁷ to R ¹⁰ independently of one another are hydrogen, phenyl, hydroxy-substituted phenyl, hydroxy, dC ₆ -acyl or dC ₆ -alkyl, where the The alkyl radical in turn can be substituted by hydroxy, dC ₆ -alkoxycarbonyl, phenyl or hydroxy-substituted phenyl,

A is a radical -O-, -S-, -SO- or -SO ₂ -,

R ^{11 is} phenyl which is optionally substituted one or more times, identically or differently, by substituents selected from the group consisting of halogen, nitro, trifluoromethyl, Ci-Cö-alkyl and d-Cö-alkoxy,

R ^{2 is} a radical of the formulas -OR ¹² or -NR ¹³ R ¹⁴ , wherein

R ^{12 is} hydrogen, dC ₆ -alkoxycarbonyl or a straight-chain, branched or cyclic, saturated or unsaturated C _j -C ₈ -hydrocarbon radical, which optionally contains one or two identical or different hetero-chain links from the group -O-, -CO- , -NH-, -N- (dC ₄ alkyl) -, -S- and -SO ₂ - and optionally by halogen, nitro, cyano, hydroxy, aryl with 6 to 10 carbon atoms or aralkyl with 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 dC ₆ -alkyl,

R ¹³ and R ¹⁴ independently of one another hydrogen, -CC ₆ alkyl or

Mean cycloalkyl having 3 to 6 carbon atoms,

R, 3 is hydrogen, amino or a radical of the formula

H ₃ CO

OCH ₃ or formyl, cyano, hydroxy-substituted C ₁ -C ₄ -alkylthio, trifluoromethyl or a straight-chain, branched or cyclic, saturated or unsaturated hydrocarbon radical having up to 8 carbon atoms, optionally with one or more, the same or different, by aryloxy 6 to 10 carbon atoms, azido, cyano, hydroxy, carboxyl, dC ₆ - alkoxycarbonyl, a 5- to 7-membered heterocyclic ring, C ₁ -C ₆ -alkylthio or dC ₆ -alkoxy (the alkylthio or alkoxy radical in turn being caused by azido , Amino or 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 Ci-Ce- alkyl, which are optionally substituted by dC ₆ - alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl, phenyl and benzyl optionally one or more times, identically or differently, by hydroxyl, carboxyl, C ₁ -C ₆ Alkyl or dC ₆ -alkoxy are substituted and or d-Cβ-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 on which they stand mean 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, and their salts.

Preference is given to compounds of the formulas I and Ia,

R ^{1 is} phenyl, furyl, thienyl, pyridyl, cyclopentyl or cyclohexyl, these ring systems optionally one or two times, identical or different by substituents, selected from the group halogen, trifluoromethyl, nitro, -SO ₂ -CF ₃ , methyl, cyano, Trifluoromethoxy, carboxyl, methoxycarbonyl or residues 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-dEL; are substituted,

R ^{2 represents} a radical of the formulas -OR ¹² or -NR ¹³ R ¹⁴ , in which

R ^{12 is} hydrogen, C ₂ -C ₄ alkenyl or dC ₄ alkyl, the alkyl radical in turn optionally being substituted by pyridyl, cyano, phenoxy, benzyl or by a radical of the formula -NR ¹⁵ R ¹⁶ , in which R ¹⁵ and R ^lδ are independently 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

H ₃ CO

OCH, or

Formyl, cyano, trifluoromethyl, cyclopropyl or dC ₄ -alkyl means, the alkyl radical in turn optionally being represented by radicals of the formulas

SO ₂ CH ₃ , -NH-CO-CH ₃ ,

-NH-CO-CF ₃ , fluorine, chlorine, dC ₃ - alkoxycarbonyl, hydroxy and / or substituted by the group - (CO) _a -NR ¹⁷ R ¹⁸ , wherein

a zero or 1,

17 1 x

R and R independently of one another are hydrogen, phenyl, benzyl or Ci

C ₄ alkyl, which are optionally substituted by dC ₃ - alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl, phenyl and benzyl independently of one another optionally optionally one or two times, identically or differently, by hydroxyl, carboxy, C ₁ -C ₃ - Alkyl or dC ₃ -alkoxy are substituted and dC -alkyl is optionally substituted by radicals of the formulas -NH-CO-CH ₃ or -NH-CO-CF ₃ , phenyl or

17 1 X R and R together with the nitrogen atom on which they stand, one

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 Cτ _. -C ₄ alkyl, and their salts.

Compounds of the formulas I and Ia are particularly preferred, embedded image in which

R ^{1 is} phenyl or thienyl, these ring systems optionally being substituted up to twice, identically or differently, by substituents selected from the group consisting of 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 d-alkyl and

R ¹³ and R ¹⁴ independently of one another denote hydrogen or methyl,

R ³ denotes hydrogen or formyl, cyano, trifluoromethyl, cyclopropyl or dC ₃ alkyl which is optionally substituted by fluorine, chlorine or hydroxyl and which in turn can be substituted up to 3 times by dC ₃ alkoxycarbonyl,

R ^{4 is} hydrogen or methyl,

D is an oxygen or sulfur atom and

R ^{5 is} hydrogen, fluorine, chlorine or -CC ₃ alkyl, and their salts.

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 methoxy, ethoxy or n-propoxy, R is hydrogen, methyl or cyclopropyl,

R ^{4 is} hydrogen,

D is an oxygen or sulfur atom and

R ^{5 is} hydrogen, fluorine or chlorine, and their salts.

Preferred such 2-heterocyclically substituted dydropyrimidines include, for example, the compounds in the following table.

Table C:

The Rr values were determined in cyclohexane / ethyl acetate (7: 3 parts by volume).

Cycloalkyl having 3 to 6 carbon atoms in the context of the invention represents cyclopropyl, cyclopentyl, cyclobutyl, cyclohexyl, preferably cyclopentyl and cyclohexyl.

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

In the context of the invention, aralkyl stands for aralkyl with preferably 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 part, the alkyl part being linear or branched can be. Preferred aralkyl radicals are benzyl and phenethyl.

Heteroaryl in the context of the invention represents 5- to 7-membered rings with preferably 1 to 3, in particular 1 or 2 identical or different heteroatoms from 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. In the context of the invention, acyl represents a straight-chain or branched acyl radical having 1 to 6, preferably 1 to 4, carbon atoms, such as, for example, acetyl and propionyl.

In the context of the invention, alkyl represents a straight-chain or branched alkyl radical having 1 to 6, preferably 1 to 4, carbon atoms, such as e.g. Methyl, ethyl, propyl, isopropyl, tert-butyl, n-pentyl and n-hexyl.

In the context of the invention, alkenyl represents a straight-chain or branched alkenyl radical having 2 to 6, preferably 3 to 5, carbon atoms, such as, for example, ethenyl,

Propenyl, isopropenyl, tert-butenyl, n-pentenyl and n-hexenyl.

In the context of the invention, alkoxy represents a straight-chain or branched alkoxy radical having 1 to 6, preferably 1 to 4, carbon atoms, such as e.g. Methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy.

In the context of the invention, alkylthio represents a straight-chain or branched alkylthio radical having 1 to 6, preferably 1 to 4, carbon atoms, such as, for example, methylthio, ethylthio and propylthio.

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

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

The compounds I and Ia can be in stereoisomeric forms, which are either like

Image and mirror image (enantiomers) or which do not behave like image and mirror image diastereomers) exist. The compounds I and Ia thus include both the enantiomers and the diastereomers and their respective Mixtures. Like the diastereomers, the racemic forms can be separated into the stereoisomerically uniform constituents in a known manner.

The compounds I and Ia can also be present as salts. In the context of the invention, physiologically acceptable salts are preferred.

Physiologically acceptable salts can be salts of the compounds I or Ia with inorganic or organic acids. Salts of inorganic acids, such as, for example, hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid, or salts of organic carboxylic or sulfonic acids, such as, for example, acetic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, or methanesulfonic acid, ethanesulfonic acid, phenylsulfonic acid , Toluenesulfonic acid or naphthalenedisulfonic acid.

Physiologically acceptable salts can also be metal or ammonium salts

Compounds I or Ia. For example, particular preference is given to Sodium, potassium, magnesium or calcium salts and ammonium salts derived from ammonia or organic amines, such as ethylamine, di- or triethylamine, di- or triethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine, lysine, ethylenediamine or 2- Phenylethylamine.

The compounds (I) or (Ia) can be prepared by

[A] aldehydes of the formula

R ^ CHO (II)

where R ^{1 has} the meaning given above,

with amidines or their hydrochlorides of the formula

in which 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 meanings given above,

with or without addition of base or acid, if appropriate in the presence of inert organic solvents, or

[B] Compounds of the formula

in which 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, with or without addition of base or acid, preferably at temperatures of 20 to 150 ° C, optionally in the presence of inert organic solvents, or

[C] Aldehydes of the formula

R ^ CHO (II)

where R ^{1 has} the meaning given above,

with compounds of the formula

R - C = C - CO-R ²

H (VI)

NH

in which R ² and R ^{3 have} the meanings given above,

and amidines of the formula (III) are reacted as described above.

The manufacturing processes can be exemplified using the following formula schemes:

[A]

[B]

[C]

For all process variants A, B and C, all inert organic solvents are suitable as solvents. These preferably include 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 dimethylformamide, dimethyl sulfoxide, acetonitrile, pyridine and hexamethylphosphoric acid triamide.

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

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

The reaction can be carried out with or without addition of base or acid, preferably in the presence of weaker acids, such as, for example, acetic acid or formic acid. The aldehydes (II) used as starting materials are known or can be prepared by methods known from the literature [cf. TD Harris and GP Roth, J. Org. Chem. 44, 146 (1979), DE-OS 2 165 260 and 2 401 665, Mijano et al., Chem. Abstr. 59, (1963), 13 929 c, E. Adler and H.-D. Becker, Chem. Scand. 15, 849 (1961), EP Papadopoulos, M. Mardin and Ch. Issidoridis, J. Org. Chem. Soc. 78

2543 (1956)].

The amidines (III) used as starting materials are known or can be prepared by methods known from the literature [cf. "Methods of 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 methods known from the literature [e.g. D. Borrmann, "Reaction 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 be prepared by methods known from the literature [cf. G. Jones, "The Knoevenagel Condition" in Organic Reactions, Vol. XV, 204 ff. (1967)].

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

The compounds I and Ia, which contain an optionally substituted oxazolyl or thiazolyl radical in the 2-position, and various processes for their preparation are known from DE-OS 198 17262 (= WO 99/54 329).

The dihydropyrimidines A act as HBV core protein inhibitors. Combinations are therefore a further subject of the invention

A) at least one HBV core protein inhibitor,

B) at least one HBV antiviral agent different from A, preferably at least one HBV polymerase inhibitor, and optionally C) at least one immunomodulator.

HBV core protein inhibitors in the sense of the invention are those non-nucleoside inhibitors which in the cell (i) at least halve the half-life of the HBV core protein or (ii) pass the binding test described below.

(i) Pulse-chase labeling of HepG2.2.15 cells and subsequent detection of the HBV core protein by immunoprecipitation:

general description

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

The amount of protein correlates with the built-in radioactivity and consequently that

Blackening of an X-ray film or the radiation intensity that can be made visible by a phosphor imager.

With the help of this technique, the influence of potential inhibitors on the half-life of the viral HBV core protein can be examined. So there is a substance an HBV-core protein inhibitor if the half-life of the HBV-core protein is at least halved; see. following test description.

Experimental Procedure

Sowing the cells:

On day 1, the HepG2.2.15 cells are used in cell culture bottles (75 cm ² ; Costar) with 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); 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 (from Sigma, Munich), aqueous sodium pyruvate solution (up to one Sodium pyruvate 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)) added. The test substance is prepared as a 50 mM stock solution in dimethyl sulfoxide (DMSO; Sigma, Munich). It is diluted in HepG2 cell culture medium and adjusted to 100 times the IC 50 concentration. 1 ml of this test substance solution is added to 19 ml of 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 the proteins:

On day 3, the culture medium is discarded and replaced by 20 ml of a medium from which the amino acids methionine and cystine are removed ("starvation medium"). The hunger 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 / 1), 5 ml aqueous glucose solution (100 g / 1), 5 ml aqueous i-inositol solution (0.2 g / 1), 5 ml aqueous L-arginine solution (12.46 g / 1) and aqueous FCS (up to an FCS concentration of the total solution of 2% v / v). The incubation with hunger 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. Then 16 ml of hunger medium are removed 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 (“pulse”). The marking solution is then removed and fed with starving 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 marking. 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 at

37 ° C and 5% CO ₂ (v / v) incubated in the incubator ("chase"). 5 minutes after adding the label stop medium, the time value to = 0 ("O-hour value") is taken. Further test times are variable in the range up to 96 hours. A sample set (treated / untreated) is taken from each further time value in the course of the "chase" in order to determine the core protein content at this point in time.

For this purpose, the medium is removed and the cells are washed once with PBS (phosphate-buffered saline without Ca / Mg **) (biochrome No. L1825). The cell lawn is then washed with 1 ml of 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]; 140 mM NaCl} lysed. (The

The term "Tris" stands for 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 and the lysate is stored at + 4 ° C.

Precipitation of the HBV core protein by specific immunoprecipitation To the lysate are added 10 ul Kanichen normal serum (Sigma 7523) and 200 ul 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 rotary wheel. The immunoprecipitates are separated by centrifugation (2 minutes; 1000 rpm) and stored at 4 ° C. The supernatant is transferred to a new Eppendorf tube.

Then 5 μl of anti-human HBV-core rabbit antiserum and 200 μl of Sepharose beads suspended in PBS (protein A-Sepharose C1-4B; Pharmacia, 17-0780-01) are added (alternatively, the complex of beads can be added and antibodies are preformed). This is followed by an incubation (2 hours at 4 ° C) on the rotary wheel. The immunoprecipitates are separated by centrifugation (2 minutes; 1000 rpm) and stored at 4 ° C. The supernatant is transferred to a 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) of the nonionic detergent © Nonident NP-40 (Boehringer Mannheim); 150 mM NaCl; 5 mM EDTA (ethylenediaminetetraacetic acid)], i.e. alternately suspended and centrifuged (2 minutes at 1000 rpm). The washed immunoprecipitates are stored at 4 ° C.

SDS page and visualization of the radioactively labeled HBV core proteins (SDS = dodecyl sulfate sodium)

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

The gel is immersed three times for 5 minutes in fixing solution (70 vol.% Ethanol; 10 vol.% Acetic acid; 20 vol.% Water). The gel is then bathed in an enhancer (Enlightning Rapid Autoradiography Enhancer NEN Research Products No. NEF-974) for 20 minutes. The gel is placed on filter paper (BioRad No. 165-0921) and covered with cellophane (BioRad No. 165-0922). This arrangement is dried for 50 minutes with the BioRad gel dryer.

The dried gel is placed on an X-ray film (Hyperfilm MP; Amersham RPN 2115H) and developed after an appropriate exposure period. Alternatively, the dried gel can be analyzed on a phosphor imager (Fujix BAS 100). The evaluation compares the half-lives of the HBV core protein in the presence and 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 naturally occurring forms of aggregation (dimers, multimers) during gel permeation chromatography depends on the choice of the gel material used. For example, the gel material used in desalination columns (e.g. Sephadex G-25; Pharmacia) may have an exclusion limit that applies to globular proteins in one

Molecular weight is 5000 daltons; ie all proteins with a molecular weight greater than 5000 Da are eluted in the exclusion volume. On the other hand, a globular protein that has a molecular weight below 5000 Da penetrates the separation material and is retained on the column. Apart from effects that are not determined by the molecular size (e.g. adsorption), the rule of thumb is: the lower the molecular weight of a molecule, the longer it will take the way through the gel bed and the later the molecule is eluted. This basic test 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. For this purpose, the test substance, which must be sufficiently below the exclusion limit, is marked (for example by replacing a hydrogen atom with tritium) and with the protein (for example the HBV core protein or its form of aggregation = "capsid") which is sufficiently above the exclusion limit must be incubated. The material is applied to the column and eluted with an elution buffer (eg 10 mM Tris pH 7.4 and 1 mM EDTA). By binding to the protein, the labeled test substance, like the free protein, is eluted in the exclusion volume. In contrast, a non-binding test substance or the excess of the test substance is eluted in the late elution volume. To demonstrate the elution behavior, individual fractions are collected and the radioactivity is determined in the scintillation counter (or by another suitable detection method).

As a rule, this method is carried out as a variant of the binding test in such a way that, for example, a labeled substance that binds to the HBV core protein (“labeled substance”) is used to find further core protein binders: the labeled one Substance is directly displaced by a test substance ("displacing substance") (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).

Carrying out the experiment Providing the column:

The Sephadex G-25 column (height of the column: 5 cm, diameter of the column: 1.5 cm) is cast according to standard procedures (manufacturer's instructions) or obtained from a manufacturer (e.g. PDIO desalting columns from Phamacia). The column is equilibrated in the desired buffer (10 mM Tris pH 7.4 and 1 mM EDTA). A sample (32 μl) consisting of 2 μl (= 64 pmol) labeled substance solution

(Specific activity of, for example, a tritiated compound is typically in the mCi / ml range), mixed with HBV core protein (10 μg = 2.7 pmol), which is in the form of the capsid, and with displacing substance (100 × molar excess, based on the labeled substance) is applied to the column applied. The equilibration buffer is used for elution. Fractions of 1 ml are collected. The entire fraction or an aliquot thereof is washed with scintillator liquid (Ultima

Gold, Packard) mixed and measured with a scintillation counter. If the test substance displaces labeled substance, less activity is measured in the fractions containing the exclusion volume than in 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 is present.

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, the binding test (ii) will be chosen because of the higher test speed; it is also suitable for screening, which can then be followed by a half-life test (i) for selected compounds. HBV core protein inhibitors in the sense of the invention are those which pass at least the half-life test (i) described above.

A particular embodiment of the invention relates to combinations of A) the above dihydropyrimidines (I) or (Ia), B) HBV polymerase inhibitors and, if appropriate, C) immunomodulators.

HBV polymerase inhibitors B in the sense of the invention are substances which are described in the endogenous polymerase assay described below, which was described by Ph. A. Furman et al. in Antimicrobial Agents and Chemotherapy, Vol. 36 (No. 12), 2688 (1992), lead to an inhibition of the formation of an HBV-DNA double strand in such a way that there is a maximum of 50% of the activity of the zero value: HBV virions from culture supernatants incorporate nucleoside 5'-triphosphates into the plus strand of HBV DNA in vitro. Using agarose gel electrophoresis, the incorporation of [α- ³² P] deoxynucleoside 5'-triphosphate into the 3.2 kb viral DNA product in the presence and absence of a substance with potential HBV polymerase inhibiting effects Properties 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 ¹ volume of an aqueous solution containing 50% by weight of 8000 polyethylene glycol and 0.6 M sodium chloride. The virions are sedimented by centrifugation at 2,500 xg / 15 minutes. The sediments are contained in 2 ml of buffer

Resuspended 0.05 M Tris-HCl (P _H 7.5) and dialyzed against the same buffer containing 100 mM potassium chloride. The samples can be frozen at -80 ° C. Each reaction mixture (100 μl) contains at least 10 ⁵ HBV virions; 50 mM Tris-HCl (p _H 7.5); 300mM potassium chloride; 50mM magnesium chloride; 0.1% ^® Nonident P-40 (non-ionic detergent from Boehringer Mannheim); 10 μM each of 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 adding 50 mM EDTA. A 10% weight volume SDS solution (containing 10 g SDS per 90 ml 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 (volume ratio 25: 24: 1), and the DNA is precipitated from the aqueous phase with ethanol. The DNA pellet is dissolved 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)) resuspended and separated by electrophoresis in an agarose gel. The gel is either dried or the nucleic acids contained therein are transferred to a membrane using the Southern transfer technique. Then the amount of the formed and labeled DNA double strand in relation to the negative control (=

Endo-Pol reaction without substance or with control substance without effect) certainly. An HBV polymerase inhibitor is present when a maximum of 50% of the activity of the negative control is present.

Preferred HBV polymerase inhibitors B) include, for example

3TC = Lamivudine =

= 4-amino-l - [(2R-cis) -2- (hydroxymethyl) -1.3-oxathiolan-5-yl] pyrimidin-2 (1H) -one, cf. EP-PS 382 526 (= US-PS 5 047 407) and WO 91/11186 (= US-PS 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 = [IS- (1.α, 3.α, 4.β)] - 2-amino-1,9-dihydro-9- [4-hydroxy-3- (hydroxymethyl) -2-methylene-cyclopentyl ] -6H-purin-6-one, cf. EP-PS 481 754 (= US-PS 5 206 244 and 5 340 816), WO 98/09964 and 99/41275;

Abacavir = (-) - (l S-cis) -4- [2-amino-6- (cyclopropylamino) -9H-purin-9-yl] -2-cyclopenten-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-l- [2- (hydroxymethyl) -1.3-oxathiolan-5-yl] pyrimidine -

2 (lH) -on, cf. WO 92/14743 (= US-PS 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;

ß-L-FDDC = 5- (6-Amino-2-fluoro-9H-purin-9-yl) -tefrahyα ^ o-2-furanmethanol, cf. WO 94/27616 (= U.S. Patents 5,627,160, 5,561,120.5,631,239 and 5,830,881);

L-FMAU = 1 - (2-deoxy-2-fluoro-ß-L-arabinofuranosyl) -5-methyl-pyrimidine-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) above dihydropyrimidines (I) or (Ia) and B) lamivudine.

Other preferred HBV antiviral agents B include e.g. Phenylpropenamide the

formula

wherein

R ¹ and R ² independently of one another denote dC -alkyl or together with the nitrogen atom on which they are located 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, -CC ₄ alkyl, -C ₇ -acyl or aralkyl and X is halogen or optionally substituted dC ₄ alkyl, and their salts.

These phenylpropenamides and processes for their preparation are known from WO 98/33501, to which reference is hereby made for the purposes of the disclosure. AT-61 is the compound of the above formula wherein X is chlorine, A 1-piperidinyl and Y and Z are each phenyl. Our own studies have shown that AT-61 is not an HBV core protein inhibitor in the sense of this invention (test see above).

Preferred immunomodulators C) include, for example, all interferons such as α, β and γ interferons, in particular also α-2a and α-2b interferons, interleukins such as interleukin-2, polypeptides such as thymosin-α-1 and thymoctonan, Imidazoquinoline derivatives such as © Levamisole, immunoglobulins and therapeutic vaccines.

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

The present invention includes pharmaceutical preparations which, in addition to non-toxic, inert pharmaceutically suitable excipients, contain one or more combinations according to the invention or which consist of a combination according to the invention, and methods for producing these preparations.

The quantitative ratio of components A, B and optionally C of the combinations according to the invention can vary within wide limits; it is preferably 5 to 500 mg A / 10 to 1000 mg B, in particular 10 to 200 mg A / 20 to 400 mg B. Component C, which may optionally be used, can be used in amounts of preferably 1 to 10 million, in particular 2 to 7 million IE (international units), applied approximately three times a week over a period of up to one year. The combinations according to the invention should generally be present in the pharmaceutical preparations listed above in a concentration of about 0.1 to 99.5, preferably about 0.5 to 95,% by weight of the total mixture.

In addition to the combinations according to the invention, the pharmaceutical preparations listed above can also contain further active pharmaceutical ingredients.

The pharmaceutical preparations listed above can be prepared in a customary manner by known methods, e.g. by mixing the active ingredient or ingredients with the carrier (s).

In general, it has proven to be advantageous in both human and veterinary medicine to use the combinations according to the invention in total amounts of about 0.5 to about 500, preferably 1 to 100 mgkg of body weight per 24 hours, optionally in the form of several individual doses Get the results you want. A single dose contains the active ingredient or active ingredients preferably in amounts of about 1 to about 80, in particular 1 to 30 mg / kg body weight. However, it may be necessary to deviate from the doses mentioned, depending on the type and body weight of the object to be treated, the type and severity of the disease, the type of preparation and administration of the drug, and the period or Interval within which the administration takes place.

The combinations according to the invention are antiviral against hepatitis B viruses

(HBV) effective by causing an extremely large reduction in intra- and / or extracellular HBV DNA. The combinations according to the invention are therefore suitable for the treatment of virus-induced diseases, in particular acute and chronic persistent viral infections of HBV. A chronic viral disease caused by HBV can lead to different degrees of severity Cause clinical pictures; As is well known, chronic hepatitis B virus infection in many cases leads to cirrhosis of the liver and / or hepatocellular carcinoma.

The indication areas for the combinations according to the invention include:

1. the treatment of acute and. chronic viral infections that can lead to infectious hepatitis, preferably the treatment of acute and chronic hepatitis B virus infections;

2. the treatment of acute and chronic HBV infections when co-infected with the hepatitis delta virus;

3. the treatment of acute and chronic HBV infections when co-infected with other viruses, such as. B. HIV or HCV, and

4. prophylactic / therapeutic treatment for transplants, such as Liver transplants.

The invention therefore also relates to the combinations defined above for combating diseases.

The invention further relates to medicaments containing at least one of the combinations defined above and optionally further pharmaceuticals

Agents.

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

Composition (mg / 1) of the lx medium made from © seromed RPMI 1640: NaCl 6000

KC1 400

Na ₂ HPO ₄ 7 H ₂ O 1512

MgSO ₄ x 7 H ₂ O 100

Ca (NO ₃ ) ₂ x 4 H ₂ O 100

D-glucose 2000

Phenol red 5

NaHCO ₃ 2000

Phenol red 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 x HC1 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 ₁ 0.005

D-Ca pantothenate 0.25

Choline chloride 3

Folic acid 1 i-inositol 35

Nicotinamide 1 p-aminobenzoic acid 1

Pyridoxine x HC1 1

Riboflavin 0.2

Thiamine x HC1 1

Composition (mg / 1) of the lx medium produced from © seromed Medium 199 with Earle's salts:

NaCl 6800

KC1 400

NaH ₂ PO ₄ x H ₂ O 140

MgSO ₄ x 7 H ₂ O 200

CaCl ₂ 200

D-glucose 1000

Phenol red 17

NaHCO ₃ 2200

DL-Alanine 50

L-Argininx HCl 70

DL-Aspartic Acid 60

L-Cysteinex HCl 0.1 L-cystine 20

L-glutamine 100

DL-glutamic acid x H ₂ O 150

glycine

L-histidine x HCl 50

L-hydroxyproline 20

DL isoleucine 10

DL-leucine 40

L-lysine x HC1 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 acetate 0.05

Fe (NO ₃ ) ₃ 50

© Tween 80 * 0.1

20

Adenine sulfate 10

Guanine x HC1 0.3

Hypoxanthine 0.3

Thymine 0.3

Uracil 0.3

Xanthine 0.3

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 x HCl 0.025

Pyridoxine x HCI 0.025

Riboflavin 0.01

Thiamine x HCI 0.01

DL-α-tocopherol phosphate Na ₂ 0.01

Vitamin A 0.1

Cholesterol 0.2

2-deoxy-D-ribose 0.5

D-Ribose 0.5 * trademark of Atlas Chemie

Medium composition (mg 1) of the MEM-lx solution made from Gibco 51091-015:

CaCl ₂ x 2 H ₂ O 264

KC1 400 MgSO ₄ x 7 H ₂ O 200

NaCl 6800

NaHCO ₃ 2200

NaH ₂ PO ₄ x2H ₂ O 158

Phenol red 10

L-histidinexHClxH ₂ O 41.92

L-isoleucine 52.46

L-LysinexHCl 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 x HC1 1.00

Riboflavin 0.10

Thiamine x HC1 1.00

Examples;

HBV in cell culture

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

The combinatorial test of the test substances was carried out by means of checkerboard titration.

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

Stock solutions of the test compounds (50 mM) were first dissolved in DMSO; further dilutions were made in growth medium. The remaining wells contained the combinations according to the invention or their individual components in the test concentrations of e.g. 5 μM to 0.01 μM, starting from A2 to Hl 1 der

96-well microtiter plate.

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

The test mixture was incubated for 8 days at 37 ° Celsius and 5% CO ₂ (v / v). On day 4, the medium was replaced by fresh inhibitor-containing medium. cytotoxicity

Before harvesting the supernatants / cell lysates to determine the antiviral effect, the HepG2.2.15 cells were examined for light-microscopic changes or by means of biochemical detection methods (e.g. Alamar blue staining or trypan blue staining) for cytotoxic changes.

Substance-induced cytotoxic or cytostatic changes in the HepG2.2.15 cells were e.g. determined by light microscopy as changes in cell morphology. Such substance-induced changes in the HepG2.2.15 cells compared to untreated cells were e.g. visible as cell lysis, vacuolization or altered cell morphology. 50% cytotoxicity ("Tox.-50") means that 50% of the cells have a morphology comparable to the corresponding cell control.

The compatibility of some combinations according to the invention was additionally tested on other host cells, e.g. HeLa cells, human primary peripheral blood cells or transformed cell lines such as H-9 cells.

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

Determination of the antiviral effect

The supernatants / cell lysates were then harvested and reduced by means of

Pressure was drawn onto 96-well dot-blot chambers covered with nylon membrane (according to the manufacturer's instructions).

In brief: After transfer of the supernatants or whole cell lysates to the nylon membrane of the blot apparatus (see above), the nucleic acids contained therein were denatured (1.5 M NaCl / 0.5 N NaOH), neutralized (3 M NaCl 0.5 M Tris Hcl, pH 7.5) and washed (2 x SSC). The DNA was then baked onto the membrane by incubating the filters at 120 ° C. for 2-4 hours.

Hybridization of the DNA

The detection of the viral DNA from the treated HepG2.2.15 cells on the nylon filters was generally carried out using non-radioactive, digoxigenin-labeled hepatitis B-specific DNA probes, which were labeled with digoxigenin according to the manufacturer's instructions, purified and used for hybidization ,

In short: the prehybidization and hybidization were carried out in 5 x SSC, 1 x blocking reagent, 0.1% N-lauroylsarcosine, 0.02% SDS and 100 μg sperm DNA of the herring. The pre-hybridization took place at 60 ° C for 30 minutes, the specific hybridization with 20 - 40 ng / ml of the digoxigenized, denatured HBV-specific DNA (14 hours, 60 ° C). The filters were then washed.

Detection of HBV DNA by digoxigenin antibodies

The digoxigenin-labeled DNA was detected immunologically according to the manufacturer's instructions.

In short: The filters were washed and prehybridized in a blocking reagent (according to the manufacturer's instructions). The mixture was then hybridized for 30 minutes with an anti-DIG antibody which was coupled with alkaline phosphatase. After a washing step, the substrate of alkaline phosphatase, CSPD, was added, 5

Incubated for minutes with the filters, then wrapped in plastic wrap and incubated for a further 15 minutes at 37 ° C. The chemiluminescence of the hepatitis B-specific DNA signals was visualized by exposing the filter to bioluminescence on an X-ray film or with a Lumi imager (incubation depending on signal strength: approx. 2 minutes to approx. 2 hours) and the degree of Measure darkness. The inhibition values were converted into% inhibition values according to the cut-off values from the internal test controls (Tab. 1). To analyze the synergistic effectiveness of the combinations, the difference values between calculated and measured inhibition values of each combination were determined (Tab. 2); see. Prichard et al.,

Antimicrob. Agents chemother. 37, 540-545 (1993).

The treatment of HBV with the combinations according to the invention has an antiviral effect better than the individual treatment; the treatment of the HepGits 2.2 virus-producing HepG2.2.15 cells with the combinations according to the invention led to a greater reduction in the intracellular viral DNA; the combination treatment is synergistically effective.

TabeUe 1

Inhibition of the combination of compound 43 from Table B with lamivudine

Table 2

Combination of compound 43 from Table B with lamivudine; Difference between expected and received inhibition

Claims

claims:

1. Combinations

A) at least one dihydropyrimidine,

B) at least one HBV antiviral agent different from A and optionally

C) at least one immunomodulator.

2. Combinations according to claim 1, wherein the dihydropyrimidine A of the formula

or their isomeric form

corresponds to what

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

or

means, where the above-mentioned ring systems optionally one or more, the same or different by substituents selected from the group halogen, trifluoromethyl, nitro, cyano, trifluoromethoxy, carboxyl, hydroxyl, dC _ö alkoxy, C ₁ -C ₆ alkoxy carbonyl and dC ₆ alkyl, where the alkyl radical in turn can be substituted by aryl having 6 to 10 carbon atoms or halogen, and the ring systems listed, if appropriate, by -SR ⁶ , -NR ⁷ R ⁸ , -CO-NR ⁹ R ¹⁰ , - SO ₂ -CF ₃ or -A-CH ₂ -R ^{U are} substituted, wherein

R ⁶ optionally halogen-substituted phenyl,

R ⁷ to R ¹⁰ independently of one another are hydrogen, phenyl, hydroxyl-substituted phenyl, hydroxyl, dC ₆ -acyl or dC ₆ -alkyl, the alkyl radical in turn being substituted by hydroxyl, dC _ö -alkoxycarbonyl, phenyl or hydroxyl-substituted phenyl can

A is a radical -O-, -S-, -SO- or -SO ₂ -,

R ^{11 is} phenyl, optionally one to more times, identically or differently, by substituents selected from the group Halogen, nitro, trifluoromethyl, -CC ₆ alkyl and -C-C ₆ -

Alkoxy, is substituted,

R ^{2 is} a radical of the formulas -XR ¹² or -NR ¹³ R ¹⁴ , in which

X is a single bond or oxygen,

R ^{12 is} hydrogen, straight-chain or branched dC ₆ -alkoxycarbonyl, a straight-chain, branched or cyclic, saturated or unsaturated C _j -C ₈ -hydrocarbon radical which optionally contains one or two identical or different hetero-chain links from the group -O-, -CO-, -NH-, -N- (Cι- C ₄ alkyl) -,

-S- or -SO ₂ - and which is optionally substituted by halogen, nitro, cyano, hydroxy, aryl having 6 to 10 carbon atoms, aralkyl having 6 to 10 carbon atoms, heteroaryl or a group of the formula -NR ¹⁵ R ¹⁶ , wherein R and R independently of one another denote hydrogen, benzyl or dC 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

mean,

R is hydrogen, amino or a radical of the formula H ₃ CO

OCH ₃ or

Formyl, cyano, hydroxy-substituted dC ₆ - alkylthio, trifluoromethyl or pyridyl or a straight-chain, branched or cyclic, saturated or unsaturated hydrocarbon radical with up to 8 carbon atoms, optionally one or more, the same or different, by aryloxy having 6 to 10 carbon atoms , Azido, halogen, cyano, hydroxy, carboxyl, dC ₆ -alkoxycarbonyl, a 5- to 7-membered heterocyclic ring, -C-C ₆ - alkylthio or Cι-C ₆ -alkoxy (the alkylthio or alkoxy radical in turn 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 having 6 to 10 carbon atoms, aralkyl having 6 to 10 carbon atoms or C ₁ -C ₆ alkyl, which are optionally substituted by dC ₆ - alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl, where Phenyl and benzyl are optionally substituted one or more times, identically or differently, by hydroxyl, carboxyl, dC ₆ alkyl or C ₁ -C ₆ alkoxy and or dC ₆ alkyl optionally by -NH-CO-CH ₃ or -NH-CO -CF _{3 is} substituted,

or

R ¹⁷ and R ¹⁸ together with the nitrogen atom on which they stand mean a morpholinyl, piperidinyl or pyrrolidinyl ring, or

R ³ optionally methoxy-substituted phenyl

or

R ² and R ³ together represent 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 and

R ⁵ pyridyl, pyrimidyl or pyrazinyl, each up to 3 times, identical or different, by halogen, hydroxy, cyano, trifluoromethyl, C ₁ -C ₆ -alkoxy, dC ₆ -alkyl, C ₁ -C ₆ -alkylthio, carbalkoxy, Cι- C ₆ - acyloxy, amino, nitro, mono- or di-dC ₆ -alkylamino can be substituted,

mean, and their salts.

3. Combinations according to claim 1, wherein the dihydropyrimidine A of the formula

or their isomeric form

corresponds to what

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

means, the ring systems listed above, if appropriate, singly or multiply, identically or differently, by substituents selected from the group halogen, trifluoromethyl, nitro, cyano, trifluoromethoxy, carboxyl, hydroxyl, dC ₆ -alkoxy, dC ₆ -alkoxycarbonyl and dC ₆ -Alkyl, are substituted, wherein the alkyl radical in turn can be substituted by aryl having 6 to 10 carbon atoms or halogen, and / or the ring systems listed, if appropriate, by groups of the formulas -SR °, -NR 7'τRι8 *, -CO-NR> 9 ^y rR> 10, -SO ₂ -CF ₃ and -A-CH ₂ -R ^{π are} substituted, wherein

R ⁶ optionally halogen-substituted phenyl,

* 7 10

R to R are independently hydrogen, phenyl, hydroxy substituted phenyl, hydroxy, Cι-C ₆ acyl or Cι-C ₆ - alkyl, where the alkyl radical in turn by hydroxy, dC ₆ alkoxy carbonyl, phenyl or hydroxy-substituted Phenyl may be substituted,

A is a radical -O-, -S-, -SO- or -SO ₂ -,

R ^{11 is} phenyl, which may be one or more, identical or different, by substituents selected from the group

Halogen, nitro, trifluoromethyl, d-Cβ-alkyl and dC _ö -

Alkoxy, is substituted,

a radical of the formulas -OR ¹² or -NR ¹³ R ¹⁴ , wherein

R ^{12 is} hydrogen, dC ₆ -alkoxycarbonyl or a straight-chain, branched or cyclic, saturated or unsaturated C _χ - C ₈ -hydrocarbon radical, which may or may not contain one or two identical or different hetero-chain ethers from the group -O-,

-CO-, -NH-, -N- (dC ₄ -alkyl) -, -S- and -SO ₂ - and optionally by halogen, nitro, cyano, hydroxy, aryl with 6 to 10 carbon atoms or aralkyl with 6 until 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 dC ₆ -alkyl,

R ¹³ and R ¹⁴ independently of one another are hydrogen, -CC ₆ -alkyl or cycloalkyl having 3 to 6 carbon atoms,

R> 3 is hydrogen, amino or a radical of the formula

H ₃ CO

OCH ₃ or

Formyl, cyano, hydroxy-substituted C _j -C ^ alkylthio, trifluoromethyl or a straight-chain, branched or cyclic, saturated or unsaturated hydrocarbon radical having up to 8 carbon atoms, optionally one or more, the same or different, by aryloxy having 6 to 10 carbon atoms , Azido, cyano, hydroxy, carboxyl, dC ₆ - alkoxycarbonyl, a 5- to 7-membered heterocyclic ring, -C-C ₆ alkylthio or dC ₆ alkoxy (where the

Alkylthio or alkoxy radical may in turn be substituted by azido, amino or hydroxyl) and / or 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 dC ₆ alkyl, which are optionally substituted by d-Ce-alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl, phenyl and benzyl optionally substituted one or more times, identically or differently by hydroxyl, carboxyl, dC _ö alkyl or Ci- C ₆ -alkoxy and / or C ₆ alkyl optionally substituted by -NH-CO-CH ₃ or -NH-CO -CF _{3 is} substituted,

or

R ¹⁷ and R ¹⁸ together with the nitrogen atom on which they stand mean a morpholinyl, piperidinyl or pyrrolidinyl ring,

R ^{4 is} hydrogen, dC ₄ alkyl, acetyl or benzoyl,

D is an oxygen or sulfur atom and

R is hydrogen, halogen or straight-chain or branched alkyl having up to 6 carbon atoms

mean, and their salts.

4. Combinations according to claim 3, wherein the dihydropyrimidine A has the formulas

and or

and / or their isomeric forms and / or their salts.

5. Combinations according to claim 3, wherein the dihydropyrimidines A of the formula

or their isomeric form

correspond to what

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

means, the ring systems listed above, if appropriate, once or more, identically or differently, by substituents selected from the group halogen, trifluoromethyl, nitro, cyano, trifluoromethoxy, carboxyl, hydroxyl, C ₁ -C ₆ -alkoxy, CrC ₆ - alkoxycarbonyl and Cι- C ₆ - alkyl, where the alkyl radical in turn can be substituted by aryl having 6 to 10 carbon atoms or halogen, and / or the ring systems listed, if appropriate, 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 ^{π are} substituted, wherein

R optionally halogen-substituted phenyl,

R ⁷ to R ¹⁰ independently of one another are hydrogen, phenyl, hydroxyl-substituted phenyl, hydroxyl, dC ₆ -acyl or dC ₆ -alkyl, the alkyl radical in turn being hydroxyl, C ₁ -C ₆ -alkoxycarbonyl, phenyl or hydroxyl-substituted phenyl can be substituted

a radical -O-, -S-, -SO- or -SO ₂ -, R ^{11 is} phenyl which is optionally substituted one or more times, identically or differently, by substituents selected from the group consisting of halogen, nitro, trifluoromethyl, dC ₆ alkyl and dC ₆ alkoxy,

mean,

R ^{2 is} a radical of the formulas -OR ¹² or -NR ¹³ R ¹⁴ , wherein

R ^{12 is} hydrogen, dC ₆ -alkoxycarbonyl or a straight-chain, branched or cyclic, saturated or unsaturated C - C ₈ -hydrocarbon radical, which may be one or two identical or different hetero-chain radicals from the group

-O-,

-CO-, -NH-, -N- (dC ₄ -alkyl) -, -S- and -SO ₂ - and optionally by halogen, nitro, cyano, hydroxy, aryl with 6 to 10 carbon atoms or aralkyl with 6 up 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 dC ₆ -alkyl,

R ¹³ and R ¹⁴ independently of one another hydrogen, dC _ö alkyl or

Mean cycloalkyl having 3 to 6 carbon atoms,

R ^{3 is} hydrogen, amino or a radical of the formula

H ₃ CO

OCH, or formyl, cyano, hydroxy-substituted C _j -C ^ alkylthio, trifluoromethyl or a straight-chain, branched or cyclic, saturated or unsaturated hydrocarbon radical having up to 8 carbon atoms, optionally one or more, the same or different, by aryloxy having 6 to 10 Carbon atoms, azido, cyano, hydroxy, carboxyl, -C-C ₆ - alkoxycarbonyl, a 5- to 7-membered heterocyclic ring, -C-C ₆ - alkylthio or Ci-C _ö alkoxy (the alkylthio or alkoxy radical in turn 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 dC ₆ -alkyl, which may be replaced by dC ₆ -alkoxycarbonyl, amino,

Hydroxyl, phenyl or benzyl are substituted, phenyl and benzyl optionally being substituted one or more times, identically or differently, by hydroxyl, carboxyl, dC ₆ -alkyl or Ci-C ₆ -alkoxy and / or Ci-Ce-alkyl optionally by - NH-CO-CH ₃ or -NH-CO-CF _{3 is} substituted,

or

R ¹⁷ and R ¹⁸ together with the nitrogen atom on which they stand mean a morpholinyl, piperidinyl or pyrrolidinyl ring,

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.

6. Combinations according to claim 3, wherein

R ^{1 is} phenyl, furyl, thienyl, pyridyl, cyclopentyl or cyclohexyl, these ring systems optionally being one or two, identical or different by substituents selected from the group halogen, trifluoromethyl, nitro,

-SO -CF ₃ , methyl, cyano, trifluoromethoxy, carboxyl, methoxycarbonyl or residues of the formulas -CO-NH-CH ₂ -C (CH ₃ ) ₃ , - CO-NH (CH ₂ ) ₂ OH ₅ -CO-NH- CH ₂ -C ₆ H ₅ , -CO-NH-C ₆ H ₅ ,

CO-NH- (pOH) -C ₆ H, -O-CH ₂ -C ₆ H ₅ or -S-ρCl-C ₆ H _{4 are} substituted,

R ^{2 represents} a radical of the formulas -OR ¹² or -NR ¹³ R ¹⁴ , in which

R ^{12 is} hydrogen, C ₂ -C ₄ alkenyl or C ₁ -C ₄ alkyl, the alkyl radical in turn optionally being 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 are 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

H ₃ CO

OCH,

or

Formyl, cyano, trifluoromethyl, cyclopropyl or C 1 -C ₄ -alkyl means, the alkyl radical in turn being optionally substituted by radicals of the formulas -SO ₂ CH ₃ , -NH-CO-CH ₃ ,

-NH-CO-CF ₃ , fluorine, chlorine, -CC ₃ alkoxycarbonyl, hydroxy and / or by 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 ₃ -C ₃ -alkoxycarbonyl, amino, hydroxyl, phenyl or benzyl, phenyl and benzyl optionally being substituted independently of one another. or substituted twice, identically or differently by hydroxy, carboxy, dC ₃ -alkyl or dC ₃ -alkoxy and dd-alkyl 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 on which they stand mean 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 d - alkyl

mean, and their salts.

7. Combinations according to claim 3, wherein

R ^{1 is} phenyl or thienyl, which are optionally substituted up to twice, identically or differently, by fluorine or chlorine,

R ² 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.

8. Combinations

A) at least one HBV core protein inhibitor,

B) at least one HBV antiviral agent different from A and optionally C) at least one immunomodulator.

9. Combinations according to claims 1 to 8, wherein component B is an HBV polymerase inhibitor.

10. Combinations according to claims 1 to 8, wherein component B is lamivudine.

11. Combinations according to claims 1 to 8, wherein component B from the compounds of the formula

wherein

R ¹ and R ² independently of one another are C ¹ -C ₄ -alkyl or, together with the nitrogen atom on which they are located, form a ring having 5 to 6 ring atoms which comprise carbon and / or oxygen,

R -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, dC ₄ alkyl, dC ₇ acyl or aralkyl and

X halogen or optionally substituted C ₁ -C ₄ alkyl mean,

and whose salts are selected.

12. Combinations according to claim 11, wherein

X is chlorine, A 1-piperidinyl and Y and Z are each phenyl.

13. Combinations according to claims 1 to 12, wherein the immunomodulator C contains interferons.

14. Combinations of A) dihydropyrimidine, B) lamivudine and optionally C) interferon.

15. A process for the preparation of the combinations according to claims 1 to 14, characterized in that components A and B are suitable

Wise combined or prepared.

16. Combinations according to claims 1 to 14 for combating diseases.

17. Medicament containing at least one combination according to claims 1 to 14 and optionally further active pharmaceutical ingredients.

18. Use of combinations of claims 1 to 14 for the manufacture of a medicament for the treatment and prophylaxis of viral diseases.

19. Use of combinations of claims 1 to 14 for the manufacture of a medicament for the treatment and prophylaxis of hepatitis B infections.