HRP960376A2 - New heterocyclic substituted pseudopeptides - Google Patents

Description

The proposed invention relates to new heterocyclic substituted pseudopeptides, methods for their production and their use as anti-retroviral agents.

Trifluoromethyl-containing pseudopeptides are described in EP 528 242 as anti-retroviral agents.

The proposed invention relates to new heterocyclic substituted pseudopeptides of the general formula (I)

[image]

where

R1 represents the rest of the formula

[image]

where

R3 and R4, the same or different, represent a heterocyclic residue of the formula

[image]

R 2 represents hydrogen or straight or branched acyl having up to 20 carbon atoms, which is substituted if necessary with carboxy, with straight or branched alkoxycarbonyl with up to 6 carbon atoms, and their salts.

The compounds according to the invention of the general formula (I) have more asymmetric carbon atoms.

A characteristic residue of the general formula (Ia)

[image]

contains 5 asymmetric carbon atoms (*), which are independently present in R or S configuration. Configurations 1(R), 2(R), 3(S), 4(S), 5(S) and 6(S) are preferred.

Amino acids in the compounds according to the invention of the general formula (I) can be present both in the L and also in the D form.

Physiologically certain salts of heterocyclic substituted pseudopeptides with trifluoromethyl-substituted 2-azabicyclooctane can be salts of the substance according to the invention with mineral acids, carboxylic or sulfonic acids. Particular preference is given, for example, to salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid6m, fumaric acid, maleic acid or benzoic acid.

Preference is given to compounds according to the invention of the general formula (I) in which

R1 represents the rest of the formula

[image]

where

R3 and R4, the same or different, represent a heterocyclic residue of the formula

[image]

R2 represents hydrogen or straight or branched acyl having up to 6 carbon atoms, and their salts.

Particular preference is given to compounds according to the invention of the general formula (I) in which

R1 represents the rest of the formula

[image]

where

R3 and R4, the same or different, represent a heterocyclic residue of the formula

[image]

R2 represents a leader or straight or branched acyl having up to 5 carbon atoms, and their salts.

Particular preference is given to compounds according to the invention of the general formula (I) in which

R 2 represents hydrogen or straight or branched acyl having up to 4 carbon atoms.

In addition, a process for the production of compounds according to the invention of the general formula (I) was found, which is characterized by the fact that

compounds of formula (II) or (III)

[image]

[image]

chemically converted with compounds of the general formula (IV)

P1-OH (IV)

where

R1 has the above meaning, if necessary with the activation of carboxylic acid, in inert solvents and in the presence of a base and/or auxiliary agent, and in the second stage, in the case that R2 ≠ H, acylation is carried out in inert solvents if necessary in the presence of a base and/or auxiliary means.

The process according to the invention can be explained, for example, by means of the following scheme of formulas:

[image]

Common inert solvents, which do not change under the reaction conditions, are suitable as solvents for all stages of the process. These primarily include organic solvents such as ether, e.g. diethylether, glycolmono- or dimethylether, dioxane or tetrahydrofuran, or hydrocarbons such as benzene, toluene, xylene, cyclohexane or petroleum fractions or halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride or dimethylsulfoxide, dimethylformamide, hexamethylphosphoric triamide acids, acetic ester, pyridine, triethylaraine or picoline. Mixtures of the mentioned solvents can also be used. Preference is given to dichloromethane, dimethylformamide or tetrahydrofuran.

As bases, depending on the individual stages of the process, conventional inorganic or organic bases are suitable. These include primarily alkali hydroxides such as sodium or potassium hydroxide, or alkali carbonates such as sodium or potassium carbonate, or alkali alcoholates such as sodium or potassium methanolate, or sodium or potassium ethanolate, or organic amines such as ethyldiisopropylamine, triethylamine, picoline, pyridine, dimethylaminopyridine or N-methylpiperidine, or amides such as sodium amide or lithium diisopropylamide, or lithium N-silylalkylamide, such as lithium N-(bis)triphenylsilylamide, or lithium alkyl such as n-butyllithium.

Bases are used in amounts from 1 mol to 10 mol, preferably from 1 mol to 3 mol in relation to 1 mol of compounds of formula (II) or (III).

Condensing agents, which can also be bases, especially if the present carboxyl group is activated as an anhydride, are suitable as auxiliary agents. Here, preference is given to conventional condensing agents such as carbodiimides, eg N,N'-diethyl-, N,N'-diisopropyl-, N,N'-dicyclohexyl-carbodiimide, N-(3-dimethyl-aminoisopropyl )-N'-ethyl-carbodiimide-hydrochloride, N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimide-p-toluenesulfonate (CMCT or morpho-CDI), or carbonyl compounds such as carbonyldiimidazole, or 1,2-oxazolium compounds as 2-ethyl-5-phenyl-1,2-oxazolium-3-sulfate or 2-tert. butyl-5-methyl-isoxazolium-perchlorate, or acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline, or propanephosphoric anhydride, or isobutylchloroformate, or benzotriazolyloxy-tri(dimethyl-amino)phosphonium-hexafluorophosphate, 1 -hydroxybenzotriazole or dimethylaminopyridine.

The reactions can be carried out both under normal pressure and also under increased or reduced pressure (for example from 0.5 to 5 bar), preferably under normal pressure.

Chemical conversion generally takes place in the temperature range from -20°C to +40°C, preferably from 0°C to room temperature.

Acylation generally takes place in the above-mentioned ethers or halogenated hydrocarbons, preferably tetrahydrofuran or methylene chloride, in the temperature range from -30°C to +50°C, preferably from -10°C to room temperature.

The organic amines and pyridines mentioned above are generally suitable as bases and/or auxiliaries for acylation. Triethylamine and dimethylaminopyridine are preferred.

Bases are used in amounts from 1 mole to 10 moles, preferably from 1 mole to 3 moles, calculated according to one mole of the alcohol in question.

Compounds of the general formula (II) are known.

The compound of formula (III) is new and can be produced, for example, by combining the compound of general formula (V)

[image]

where

W representing an amino protecting group, preferably tert.butoxycarbonyl, is first converted by an epoxidation reaction, if necessary using a base or a phase transfer catalyst, into a compound of the general formula (VI)

[image]

where

W has the above meaning, and is finally chemically converted with 3-trifluoromethyl-2-azabicyclo[3. 3. 0]octane of formula (VII)

[image]

in solvents, and the protecting group is removed by known methods.

Common organic solvents that do not change under the reaction conditions are suitable as solvents. These include primarily organic solvents such as alcohols, e.g. methanol, ethanol or n-propanol, ether, e.g. diethylether, glycolmono- or dimethylether, dioxane or tetrahydrofuran, or hydrocarbons such as benzene, toluene, xylene, cyclohexane or petroleum fractions or halogenated hydrocarbons such as methylene chloride, dichloroethane (DCE), chloroform, carbon tetrachloride, or dimethylsulfoxide, dimethylformamide, hexamethylphosphoric acid triamide, acetic ester, pyridine, triethylamine, or picoline. Mixtures of the mentioned solvents can also be used. Particular preference is given to dichloromethane, dichloroethane, dimethylformamide or n-propanol.

Suitable reagents for epoxidation are compounds known from the literature, such as m-chloroperpebzoic acid, magnesium monoperoxyphthalate, dimethyldioxirane or methyl(trifluoromethyl)dioxirane. Preferred are m-chloro-perbenzoic acid and magnesium monoperoxyphthalate [compare P. Brongham et al., Synthesis (1987), 1015; W. Adam et al., J. Org. Chem. 52, 2800 (1987) and R. Curci et al. J. Org. Chem. 53, 3890 (1988)].

If the epoxidation is carried out using a phase transfer catalyst, organic ammonium chlorides or bromides such as benzyltriethylammonium chloride or bromide, methyltrioctylammonium chloride, tetrabutylammonium bromide, trimethylammonium chloride (Aliquat 336) are used as auxiliary substances. Benzyltriethylammonium chloride and bromide are preferred.

Epoxidation is carried out in the temperature range from -10°C to +904C, preferably from 0°C to +60+C.

The reactions can be carried out both under normal pressure and also under increased or reduced pressure (for example from 0.5 to 5 bar), preferably under normal pressure.

Compounds of general formula (IV) are known or can be prepared by conventional methods.

The compounds of the general formulas (V) and (VI) are mostly known (compare EP 528 242).

The compounds of the general formula (VII) are new and can be produced, for example, by fluorinating 2-azabicyclo[3.3.0]octane-3-carboxylic acid in the respective configuration with HF and SF4.

Surprisingly, it has been found that compounds of general formula (I) exhibit remarkably strong activity against retroviruses. This was confirmed by testing the HIV-specific protease enzyme.

The results of the examples below were obtained according to the HlV-assay system described in the following literature [compare Hansen, J., Billich, S., Schulze, T., Sukrov, S. and Mölling, K. (1988), EMBO Journal, Vol, 7, no. 6 p. 17 85-17 91]. Purified HlV-protease was incubated with a synthetic peptide, which imitates the intersection in the gag-precursor protein and represents the in vivo intersection of HIV-protease. The resulting cleavage products of the synthetic peptide were analyzed using reverse phase high performance liquid chromatography (RP-HPLC). The specified IC50 values refer to the concentration of the substance which, under the above test conditions, causes a 50% suppression of protease activity.

Enzyme assay, HIV-1

Table I

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In addition, the compounds according to the invention show activity in cell cultures infected with lentivirus. This can be demonstrated on the example of the HIV virus.

HIV infection in cell culture

The HIV test was performed with unknown modifications according to the method of Pauwels et al. [compare Journal of Virological Methods 20, (1988), 309-321].

Normal human blood lymphocytes (PBL's) were concentrated over Ficoll-Hypaque and stimulated in RPMI 1640, 20% calf serum with phytohemagglutinin (90 µg/ml) and interleukin-2 (40 U/ml). For infection with infectious HIV PBLs were pelleted and the cell pellet was finally suspended in 1 ml of HIV-virus absorption solution and incubated for 1 hour at 37°C.

The viral absorption solution was centrifuged and the infected cell pellet was taken up in the growth medium, so that 1x105 cells per ml were prepared. In this way, the infected cells were pipetted at 1x104 cells/well into a microtiter plate with 96 wells.

The first vertical row contained only growth substrate and cells that were not infected but were otherwise treated exactly as described above (control cells). The second vertical array contained only HIV-infected cells (viral control cells) in the growth medium. The remaining wells contained the compounds according to the invention in different concentrations starting from the wells of the 3rd vertical row of the microtiter plate, from which the test substances were diluted 210-fold in each subsequent row.

The test substances were incubated at 37°C until the formation of syncytia typical of HIV in the untreated viral control (between the third and sixth day after infection), and then microscopic evaluation was performed. Under these test conditions, approximately 20 syncytia were formed in the viral control, while untreated control cells showed none.

IC50 values were determined as the concentration of treated and infected cells, in which 50% (approx. 10 syncytia) of virally induced syncytia were suppressed by treatment with the compound according to the invention.

It has now been found that the compounds of the invention protect HIV-infected cells from virus-induced cell destruction.

Cell culture testing, PBL

Table II

[image]

The compounds according to the invention represent a valuable active substance for the treatment and prophylaxis of diseases caused by retroviruses in human medicine and veterinary medicine.

The areas of indication in human medicine can be mentioned, for example:

1.) Treatment and prophylaxis of human retroviral infections.

2.) Treatment or prophylaxis of diseases caused by HIV I (human immunodeficiency virus; previously called HTLV III/LAV) and diseases caused by HIV II (AIDS) and their accompanying conditions such as ARC (AIDS related complex) and LAS (lymphadenopathy syndrome), and immune weakness and encephalopathy caused by that virus.

3.) Treatment or prophylaxis of HTLV-I or HTLV-II infection.

4.) treatment or prophylaxis of the AIDS-carrier condition.

Indications in veterinary medicine include, for example, infections with:

a) Maedivisna (in sheep and goats),

b) progressive pneumovirus (PPV) (in sheep and goats),

c) caprine arthritis encephalitis virus (in sheep and goats),

d) Zwoegerziekt virus (in sheep),

e) infectious anemia virus (horse),

f) infections caused by feline leukemia virus,

g) infections caused by feline immunodeficiency virus (FIV),

h) infections caused by simian immunodeficiency virus (SIV).

First of all, it is the area of indications in human medicine mentioned above under points 2, 3 and 4.

The proposed invention includes pharmaceutical preparations, which, in addition to a non-toxic, inert pharmaceutically suitable carrier, contain one or more compounds of formula (I), or which consist of one or more active substances of formula (I), and methods for the production of these preparations.

The active substances of formula (I) in the above-mentioned pharmaceutical preparations must be present in a concentration of approx. 0.1 to 99.5 wt. %, especially from approx. 0.5 to 95 wt. % of the total mixture.

In addition to the compounds of formula (I), the above-mentioned pharmaceutical preparations may also contain further pharmaceutical active substances.

The production of the above-mentioned pharmaceutical preparations is carried out in the usual way by known methods, for example by mixing one or more active substances with one or more carriers.

In order to achieve the desired result, both in human medicine and also in veterinary medicine, it has generally been shown to be useful to give one or more active substances in a total amount of approx. 0.5 to approx. 500, preferably 1 to 100 mg/kg of body weight every 24 hours, if necessary in the form of several individual doses. A single dose contains one or more active substances in an amount of approx. 1 to approx. 80, especially 1 to 30 mg/kg of body weight. However, it may still be necessary to deviate from the stated dosage, depending on the type and body weight of the treated patient, the type and severity of the disease, the type of preparation and application of the drug, and the time interval, or interval, in which the drug is administered.

The compounds according to the invention are enzyme inhibitors and as such can be used for all purposes where enzyme inhibitors are required. Examples of this include use for labeling in affinity chromatography, use as an aid to elucidate enzyme structures and reaction mechanisms, and use as a diagnostic reagent.

Example 1

(2S,3S)-3-(tetrahydrofuran-3-yl-oxycarbonyl-L-asparaginyl)-amino-1-{(S,S,S)-3-trifluoromethyl-2-azabicyclo[3.3.0]octane-2 -yl}-2-hydroxy-4-phenylbutane

[image]

0.1 g (0.22 mmol) (2R,3S)-3-L-asparaginyl)amino-1-{(S,S,S)-3-trifluoromethyl-2-azabicyclo[3.3.0]octane-2 -yl}-2-hydroxy-4-phenylbutane and 0.11 g (0.44 mmol) of tetrahydrofuran-3-yl-4-nitrophenylcarbonate (analogous to Daniel F. Veber et al., J. Org. Chem. , Vol. 42, No. 20, 1977, pp. 3286-8) was dissolved in 3 ml of THF and stirred overnight at room temperature. Finally, it is evaporated to dryness and the residue is separated by column chromatography (silica gel 60, dichloromethane/methanol = 100/5, Rf = 0.4).

Yield: 120 mg (96.0% of theoretical), colorless foam.

Analogous to the regulation of example 1, the compounds listed in table 1 were produced.

Table 1.

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Example 5

(2R,3S)-3-(tetrahydrofuran-3-yl-oxycarbonyl)-amino-1-{(S,S,S)-3-trifluoromethyl-2-azabicyclo[3.3.0]octan-2-yl} -2-hydroxy-4-phenylbutane

[image]

0.1 g (0.29 mmol) (2R, 3S) -3-amino-3-{ (S, S, S) -3-trifluoromethyl-2-azabicyclo[3.3.0]octan-2-yl}- 2-hydroxy-4-phenylbutane and 0.11 g (0.44 mmol) of tetrahydrofuran-3-yl-4-nitrophenylcarbonate (analogously according to Daniel F. Veber et al., J. Org. Chem., Vol. 42, no. 20, pp. 3286-8) was dissolved in 3 ml of THF and stirred overnight at room temperature. Finally, it is evaporated to dryness and the residue is separated by column chromatography (silica gel 60, dichloromethane/ethyl acetic acid = 4/1). Rf = 0.5 (dichloromethane/ethyl ester = 1/1)

Yield: 50 mg (37.5% of theoretical), colorless foam.

Analogous to the regulation of example 5, the compounds listed in table 2 were produced.

Table 2.

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Example 9

(2S,3S)-3-(tetrahydrofuran-3-yl-oxycarbonyl-L-asparaginyl)amino-1-{(S,S,S)-3-trifluoromethyl-2-azabicyclo[3.3.0]octan-2- yl}-2-acetoxy-4-phenylbutane

[image]

50 mg (0.0876 mmol) of the compound from example 1, 25 mg (0.035 ml) of triethylamine and 13 mg (0.13 mmol) of acetic anhydride are dissolved in an argon atmosphere in 2 ml of dry tetrahydrofuran (THF), mixed with a little (at tip of a knife) of dimethylaminopytidine and mixed overnight.

The mixture is evaporated to dryness under vacuum and mixed with 5 ml of water and 15 ml of ethyl acetic acid. The organic phase is separated, dried with sodium sulfate and separated by column chromatography (silica gel 60, eluent: dichloromethane/ethyl acetate = 4/1).

Colorless crystals, melting point 191°C; Rf = 0.4 (dichloromethane/ethyl acetate = 4/1), yield: 35 mg (65.2% of theoretical).

Example 10

(2S, 3S) -3-[tetrahydrofuran-(3S)-3-yl-oxycarbonyl]-amino-1-{ (S, S, S) -3-trifluoromethyl-2-azabicyclo[3.3.0]octane-2 -yl}-2-isobutyroxyoxy-4-phenylbutane

[image]

Analogous to the procedure of Example 9, the title compound was prepared from 50 mg (0.11 mmol) of the compound from Example 6, 20 mg (0.027 ml/0.2 mmol) of triethylamine and 15 mg (0.14 mmol) of butyric anhydride with a small amount (at the tip of the knife) dimethylaminopyridine.

Colorless foam; Rf = 0.6 (dichloromethane/ethyl acetate = 4/1), yield: 40 mg (69.3% of theoretical).

Claims (7)

1. Heterocyclic substituted pseudopeptides of general formula (I) [image] indicated by that R1 represents the rest of the formula [image] where R3 and R4, the same or different, represent a heterocyclic residue of the formula [image] R2 represents hydrogen or straight or branched acyl having up to 20 carbon atoms, which is optionally substituted with carboxy, with linear or branched alkoxycarbonyl having up to 6 carbon atoms, and their salts. 2. Compounds of the general formula (I) according to claim 1, characterized in that R1 represents the rest of the formula [image] where R3 and R4, the same or different, represent a heterocyclic residue of the formula [image] R2 represents hydrogen or straight or branched acyl having up to 6 carbon atoms, and their salts. 3. Compounds of the general formula (I) according to claim 1, characterized in that R1 represents the rest of the formula [image] where R3 and R4, the same or different, represent a heterocyclic residue of the formula [image] R2 represents hydrogen or straight or branched acyl having up to 5 carbon atoms, and their salts. 4. Compounds of the general formula (I) according to claim 1, characterized in that R 2 represents hydrogen or straight or branched acyl having up to 4 carbon atoms. 5. Process for the production of compounds of the general formula (I) according to claim 1, characterized in that the compounds of the formula (II) or (III) [image] chemically converted with compounds of the general formula (IV) R1-OH (IV) where R1 has the above meaning, if necessary with the activation of carboxylic acid, in inert solvents and in the presence of a base and/or auxiliary agent, and in the second stage, in the case that R2 ≠ H, acylation is carried out in inert solvents if necessary in the presence of a base and/ or auxiliary means. 6. Medicine, characterized in that it contains one or more compounds from claims 1 to 4. 7. Use of compounds from claims 1 to 4, characterized in that they are used for the production of antiretroviral drugs.