NO963598L - Novel heterocyclic substituted pseudopeptides - Google Patents

Description

The present invention relates to new heterocyclic substituted pseudopeptides, a method for their preparation and their use as anti-retroviral agents.

Trifluoromethyl-containing pseudopeptides are described in the public domain EP 528242 as anti-retroviral agents.

The present invention relates to new heterocyclic substituted pseudopeptides of general formula fl)

in which

R.<1> stands for a remainder with the formula

in which

R.3 and R.<4> are the same or different and mean a heterocyclic residue of the formula

R<2> stands for hydrogen or for straight-chain or branched acyl with up to 20 carbon atoms, which is optionally substituted with carboxy, with straight-chain or branched alkoxycarbonyl with up to 6 carbon atoms,

and their salts.

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

The representative residue of general formula (Ia)

has 5 asymmetric carbon atoms (<*>), which can independently exist in R or S configuration. Preferred are the configurations 1(R), 2(R). 3(S), 4(S), 5(S) and 6S). The amino acids used in the compounds of general formula (I) according to the invention can be present both in the L and in the D form. Physiologically acceptable salts of the heterocyclic substituted pseudopeptides with trifluoromethyl substituted 2-azabiscyclooctane can be salts of the substances according to the invention with mineral acids, carboxylic acids or sulphonic acids. Especially preferred are e.g. salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phorsphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid. Preferred are the compounds of general formula (I) according to the invention, in which Ri stands for a residue with the formula; ; wherein;R) and R.<4>are the same or different and mean a heterocyclic residue of the formula ; ; R<2> stands for hydrogen or for straight-chain or branched acyl with up to 6 carbon atoms, and their salts. Particularly preferred are the compounds according to the invention with general formula (I), in which Ri stands for a residue with the formula; ; wherein; R<3> and R<4> are the same or different and represent a heterocyclic residue of the formula; R- stands for hydrogen or for straight-chain or branched acyl with up to 5 carbon atoms, and their salts. Particularly preferred are the compounds of general formula (I) according to the invention, in which R<2> stands for hydrogen or for straight-chain or branched acyl with up to 4 carbon atoms. Furthermore, a method has been found for the preparation of the compounds of general formula (I) according to the invention, which is characterized by reacting compounds of formula (II) or (III) with compounds of general formula (IV). ; wherein;R.<1>has the meaning given above.;optionally during prior activation of the carboxylic acid, in inert solvents and in the presence of a base and/or an auxiliary, and in a second step, in the case of R.2 * H, carries out an acylation in inert solvents, optionally in the presence of a base and/or an auxiliary agent.

The method according to the invention can, for example, be illustrated with the help of the following formula:

Suitable solvents for all process steps are the usual inert solvents which do not change under the reaction conditions. These preferably include organic solvents such as ether, e.g. diethyl ether, glycol mono- or

-dimethyl ether, 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 acid triamide, acetic ester, pyridine, triethylamine or picoline. Likewise, it is possible to use mixtures of the mentioned solvents. Particularly preferred are dichloromethane, dimethylformamide or tetrahydrofuran.

Depending on the individual method steps, the usual inorganic or organic bases are suitable as bases. These preferably include 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, pyridines, dimethylaminopyridine or N-methylpiperidine, or amides, such as natirumamide or lithium diisopropylamide, or lithium N-silylalkyl amides, such as lithium N-(bis)triphenylsilylamide, or lithium alkyls, such as n-butyllithium.

The base is used in an amount of 1 mol to 10 mol, preferably of 1 mol to 3 mol, based on 1 mol of the compounds of formula (II) or (III).

Condensing agents, which can also be bases, are particularly suitable as auxiliaries, especially when the carboxyl group is activated as an anhydride. Preferred here are the usual condensation agents such as carbodiimides, e.g. N,N'-diethyl-, N,N'-diisopropyl-, N,N'-dicyclohexylcarbodiimide, N-(3-dimethylaminoisopropyl)-N'-ethylcarbodiimide hydrochloride, N-cyclohexyl-N'-(2- morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate (CMCT or morpho-CDI), or carbonyl compounds, such as carbonyl-diimidazole or 1,2-oxazolium compounds, such 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 propanephosphonic anhydride or isobutylchloroformate, or benzotriazolyloxy-tri(dimethylamino)phosphonium hexafluorophosphate, 1 -hydroxybenzotriazole or dimethylaminopyridine.

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

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

The acylation generally takes place in one of the above-mentioned ethers or halohydrocarbons, preferably tetrahydrofuran or methylene chloride. in a temperature range from

-30°C to +50°C, preferably from -10°C to room temperature.

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

The base is used in an amount from 1 mol to 10 mol, preferably from 1 mol to 3 mol, based on 1 mol of the alcohol in question.

The compound of general formula (II) is known.

The compound with general formula (III) is new and can, for example, be prepared by transferring compounds with general formula (V)

in which

W stands for an amino protecting group, preferably tert.butoxycarbonyl,

first with an oxidation reaction, optionally with the aid of a base, or a phase transfer catalyst. to the compounds of general formula (VI),

in which

W has the above meaning.

and then reacting with 3-trifluoromethyl-2-azabicyclo[3,3.0]octane of formula (VII),

in solvents and cleave off the protecting group by conventional methods.

Suitable solvents are the usual organic solvents which do not change under the reaction conditions. These preferably include organic solvents, such as alcohols, e.g. methanol, ethanol or n-propanol, ethers, e.g. diethyl ether, glycol mono- or -dimethyl ether, dioxane or tetrahydrofuran, or hydrocarbons, such as benzene, toluene, xylene, cyclohexane or crude oil fractions or halogenated hydrocarbons, such as methylene chloride, dichloroethane (DCE), chloroform, carbon tetrachloride or dimethyl sulfoxide, dimethylformamide, hexamethylphosphoric acid triamide, acetic ester, pyridine, triethylamine or picoline. Likewise, it is possible to use mixtures of the mentioned solvents. Particularly preferred are dichloromethane, dichloroethane, dimethylformamide or n-propanol.

Suitable reagents for the epoxidation are the compounds known from the literature, such as, for example, m-chloroperbenzoic acid, magnesium monoperoxyphthalate, dimethyldioxirane or methyl(trifluoromethyl)dioxirane. Preferred are m-chloroperbenzoic acid and magnesium monoperoxyphthalate [cf. P. Brongham et al., Synthesis (1987), 1015; W. Adam et al., J. Org. Chem. 52, 2800 (1987) and R. Curci et al... Org. Chem. 51, 3890

(1988)].

If the epoxidation is carried out with the help of a phase transfer catalyst, organic ammonium chlorides or bromides are used as auxiliaries, such as for example benzyltriethylammonium chloride or bromide, methyltrioctylammonium chloride, tetrabutylammonium bromide, tricaprylmethylammonium chloride ("Aliquat 336") - Benzyltriethylammonium chloride and bromide are preferred.

The epoxidation is carried out in a temperature range from -10°C to +90°C, preferably from 0°C to +60°C.

The reaction can be carried out at normal pressure as well as at elevated or reduced pressure (for example 0.5 to 5 bar), preferably at normal pressure.

The compounds of general formula (IV) are known in and of themselves, or can be prepared by usual methods.

The compounds with general formulas (V) or (VI) are for the most part known [cf. EP 528242].

The compound with general formula (VII) is new and can, for example, be prepared by fluorinating 2-azabicyclo[3,3,0]octane-3-carboxylic acid in the relevant configuration with HF and SF4.

It was surprisingly found that the compounds of general formula (I) have an unfavorable strong effect against retroviruses. This is substantiated with an HIV-specific protease enzyme test.

The results in the examples given below were determined by the HIV test system described in the following literature reference [cf. Hansen, J., Billich, S., Schulze, T., Sukrow, S. and Molling, K. (1988), EMBO Journal, Vol 7, No 6, pp 1785-1791]: Purified HIV protease was incubated with synthetic peptide, which mimics a cut site in Gaf precursor protein and constitutes an in vivo cleavage site of HIV protease. The resulting cleavage products of the synthetic peptide were analyzed by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The indicated ICsQ values refer to the substance concentration which, under the experimental conditions indicated above, causes a 50% inhibition of the protease activity.

Enzyme analysis. HIV-1

Moreover, the compounds according to the invention showed efficacy in Lentivirus-infected cell cultures. This could, for example, be attributed to the HIV virus.

HIV infection in cell culture

The HIV test was carried out with few modifications according to the method according to Pauwels et al. [cf. Journal of Virological Methods 2Q, (1988), 309-321].

Normal human blood lymphocytes (PBLs) were enriched via Ficoll-Hypaque and stimulated in RPM1 1640, 20% fetal calf serum with phytohemagglutinin (90 µg/ml) and interleukin-2 (40 U/ml). For infection with the infectious HIV, PBLs were pelleted and the cell pellet was then suspended in 1 ml of HIV virus adsorption solution and incubated for 1 hour at 37°C.

The virus adsorption solution was centrifuged and the infected cell pellet taken up in growth medium, so that 1 x 10^ cells per cell were set. ml. The cells infected in this way were pipetted at 1 x 10 5 cells/well into the wells of 96-microtiter plates.

The dry vertical row of the microtiter plate contained only growth medium and cells that were not infected, but otherwise treated exactly as described above (cell control). The second vertical row of the microtiter plate contained only HIV-infected cells (virus control) in growth medium. The other wells contained the compounds according to the invention in different concentrations, starting from the wells in the 3rd vertical row of the microtiter plates, from which the test substances were diluted in 2nd steps 2<10> times.

The test mixtures were incubated at 37°C until, in the untreated virus control, syncytia formation typical of HIV appeared (between days 3 and 6 after infection), which was then assessed microscopically. In the untreated virus control, under these experimental conditions, approx. 20 syncytia, while the untreated cell control did not show any syncytia.

The IC5Q values were determined as the concentration of the treated and infected cells at which 50% (about 10 syncytia) of the virus-induced syncytia were suppressed by treatment with the compound according to the invention.

It was now found that the compounds according to the invention protect HIV-infected cells against the virus-induced cell destruction.

Cell culture analysis. PBL

The compounds according to the invention constitute valuable active substances for the treatment and prophylaxis of diseases caused by retroviruses. within human and veterinary medicine.

Examples of indication areas within human medicine include:

1. ) Treatment and prophylaxis of human retrovirus infections.

2. ) Treatment or prophylaxis of HIV I (human immunodeficiency virus; previously designated HTLV III/LAV) and HIV II caused diseases (AIDS) and the associated stages such as ARC (AlDS-related complex) and LAS (lymphadenopathy syndrome) as well as immunodeficiencies and encephalopathies caused by this virus.

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

4. ) Treatment or prophylaxis of the AlDS carrier state (AIDS and transmits the state).

Examples of indications within veterinary medicine include:

Infections with

a) maedi-visna (in sheep and goats)

b) progressive pneumonia virus (PPV) (in sheep and goats)

c) caprine arthritis cencephalitis virus (in sheep and goats)

d) zwoegersiekte virus (in sheep)

e) infectious viruses in anemia (horse)

f) infections caused by feline leukemia virus

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

h) infections caused by simian immunodeficiency virus (SIV)

Preferred from the indication area within human medicine are those listed above

points 2, 3 and 4.

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

The active substances of formula (I) must be present in the above-mentioned pharmaceutical preparations in a concentration of approx. 0.1 to 99.5% by weight, preferably of approx. 0.5 to 95% by weight 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 takes place in the usual way by known methods, e.g. by mixing the active substance(s) with the carrier substances.

In general, both in human and veterinary medicine it has proven advantageous to administer the active substance(s) according to the invention in total amounts of approx. 0.5 to approx. 500, preferably 1 to 100 mg/kg body weight within 24 hours, possibly in the form of several single injections, to achieve the desired results. A single supply contains the active substance(s), preferably in amounts from approx. 1 to approx. 80, especially 1 to 30 mg/kg body weight. However, it may be necessary to deviate from the dosages mentioned, more precisely depending on the type and body weight of the treatment object, the type and degree of the disease, the type of preparation and the supply of the drug, as well as the time period or interval during which the administration takes place.

The compounds according to the invention are enzyme inhibitors and as such can be used for all purposes for which enzyme inhibitors are usable. For example, use as a label for affinity chromatography, use as an aid for elucidating enzyme structures and reaction mechanisms, and use as a reagent for diagnostics should be mentioned here.

Example 1

(2S,3S)-3-(tetrahydrofuran-3-yloxycarbonyl-L-asparaginyl)amino-1-{(S,S,S)-3-trifluoromethyl-2-azabicyclo[3,3,0]octan-2- yl}-2-hydroxy-4-phenylbutane

0.1 g (0.22 mmol) (2R,3S)-3-(L-asparaginyl)amino-1-{(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-nitrophenyl carbonate (analog Daniel F. Veber et al., J. Org. Chem. , vol. 42, no. 20, 1977, pages 3286-8) is dissolved in 3 ml of THF and stirred under an argon atmosphere overnight at RT. It is then 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 theory) colorless foam.

Analogous to the procedure in example 1, the compounds listed in table 1 are prepared:

Example 5 (2R,3S)-3-(tetrahydromran-3-yl-oxycarbonyl)amino-1-{(S3,S)-3-trifluoromethyl-2-azabicyclo[3,3,0]octan-2-yl} -2-hydroxy-4-phenylbutane

0.1 g (0.29 mmol) (2R,3S)-3-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) tetrahydrofuran-3-yl-4-nitrophenyl carbonate (analog Daniel F. Veber et al., J. Org. Chem., vol. 42, no. 20, page 3286-8) dissolve in 3 ml abs. THF and stirred under argon overnight at RT. It is then evaporated to dryness and the residue is separated by column chromatography ("Silica gel 60"; dichloromethane/acetic acid ethyl ester = 4/1).

Rf =0.5 (dichloromethane/acetic ester = 1/1)

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

Analogous to the procedure in example 5, the compounds listed in table 2 are prepared.

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

The mixture is evaporated in vacuo to dryness and stirred with 5 ml of water and 15 ml of acetic acid ethyl ester. 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, Fp = 191°C; Rf = 0.2 (dichloromethane/ethyl acetate = 4/1), yield: 35 mg (65.2% of theory).

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-isobutyroxy-4-phenylbutane

Analogously to the procedure in example 9, the compound in the title is prepared from 50 mg (0.11 mmol) of the compound from example 6, 20 mg (0.027 ml/0.2 mmol) triethylamine and 15 mg (0.14 mmol) butyric anhydride with spatula tip dimethylaminopyridine.

Colorless foam; Rf = 0.6 (dichloromethane/ethyl acetate 4:1)

Yield: 40 mg (69.3% of theory).

Claims (6)

1. Heterocyclic substituted pseudopeptides, characterized by the general formula (I) in which R. <1> stands for a remainder with the formula in which R <3> and R <4> are the same or different and mean a heterocyclic residue with the formula R <2> stands for hydrogen or for straight-chain or branched acyl with up to 20 carbons atoms, which are optionally substituted with carboxy, with straight-chain or branched alkoxycarbonyl with up to 6 carbon atoms, and their salts. 2. Compounds with general formula (I) according to claim 1, characterized in that P <J> stands for a residue with the formula in whichR. <3> and R <4> are the same or different and mean a heterocyclic residue of the formula R <2> stands for hydrogen or for straight-chain or branched acyl with up to 6 carbons atoms, and their salts. 3. Compounds of general formula (I) according to claim 1, characterized in that Ri stands for a remainder with the formula in whichR. <3> and R <1*> are the same or different and mean a heterocyclic residue with the formula ;R <2> stands for hydrogen or for straight-chain or branched acyl with up to 5 carbons atoms, and their salts.; 4. Compounds with general formula (I) according to claim 1, characterized in that R <2> stands for hydrogen or for straight-chain or branched acyl with up to 4 carbon atoms.; 5. Process for the preparation of compounds of general formula (I) according to claim 1, characterized in that one reacts compounds of formulas (II) or (III) with compounds of general formula (IV), wherein Ri has the meaning given in claim 1, optionally during prior activation of the carboxylic acid. in inert solvents and in the presence of a base and/or an auxiliary agent, and in another step, in the case that R.2 * H, an acylation is carried out in inert solvents, possibly in the presence of a base and/or an auxiliary agent. 6. Medicinal product, characterized in that it contains one or more compounds according to claims 1 to 4.